Down-regulation of Bcl-2 and Bcl-xL expression with bispecific antisense treatment in glioblastoma cell lines induce cell death

The functions of the antiapoptotic proteins Bcl-2 and Bcl-xL were examined in glioblastoma cells. Expression of both Bcl-2 and Bcl-xL were found to be elevated in protein lysates from seven early passage cell lines derived from human glioblastoma tumors compared with non-neoplastic glial cells. Down-regulation of both bcl-2 and bcl-xL expression in glioblastoma cell lines U87 and NS008 with bcl-2/bcl-xL bispecific antisense oligonucleotide resulted in spontaneous cell death. The mechanism of cell death was partially caspase-dependent. Executioner caspase 6 and caspase 7, but not caspase 3, were involved in apoptosis induced by bcl-2/bcl-xL antisense treatment. Interestingly, western blots failed to demonstrate expression of caspase 3 in two of the seven glioblastoma cell lines examined. The data support the hypothesis that Bcl-2 and Bcl-xL are important in preventing cell death in glioblastoma cells. It also suggests that there are functional pathways capable of successful completion of caspase-dependent cell death in gliomas. These findings support a potential role of bcl-2/bcl-xL bispecifc antisense oligonucleotide therapy as a treatment strategy to enhance caspase-dependent cell death in patients with glioblastoma.     

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.     

Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies

Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.     

Towards the next generation of dual Bcl-2/Bcl-xL inhibitors

Structural modifications of the left-hand side of compound 1 were identified which retained or improved potent binding to Bcl-2 and Bcl-x_L in in vitro biochemical assays and had strong activity in an RS4;11 apoptotic cellular assay. For example, sulfoxide diastereomer 13 maintained good binding affinity and comparable cellular potency to 1 while improving aqueous solubility. The corresponding diastereomer (14) was significantly less potent in the cell, and docking studies suggest that this is due to a stereochemical preference for the R_S versus S_S sulfoxide. Appending a dimethylaminoethoxy side chain (27) adjacent to the benzylic position of the biphenyl moiety of 1 improved cellular activity by approximately three-fold, and this activity was corroborated in cell lines overexpressing Bcl-2 and Bcl-x_L.     

Bcl-2-regulated cell death signalling in the prevention of autoimmunity

Cell death mediated through the intrinsic, Bcl-2-regulated mitochondrial apoptosis signalling pathway is critical for lymphocyte development and the establishment of central and maintenance of peripheral tolerance. Defects in Bcl-2-regulated cell death signalling have been reported to cause or correlate with autoimmunity in mice and men. This review focuses on the role of Bcl-2 family proteins implicated in the development of autoimmune disorders and their potential as targets for therapeutic intervention.     

Differential regulation of nuclear and mitochondrial Bcl-2 in T cell apoptosis

Activated T cells require anti-apoptotic cytokines for their survival. The anti-apoptotic effects of these factors are mediated by their influence on the balance of expression and localisation of pro- and anti-apoptotic members of the Bcl-2 family. Among the anti-apoptotic Bcl-2 family members, the expression level of Bcl-2 itself and its interaction with the pro-apoptotic protein Bim are now regarded as crucial for the regulation of survival in activated T cells. We studied the changes in Bcl-2 levels and its subcellular distribution in relation to mitochondrial depolarisation and caspase activation in survival factor deprived T cells. Intriguingly, the total Bcl-2 level appeared to remain stable, even after caspase 3 activation indicated entry into the execution phase of apoptosis. However, cell fractionation experiments showed that while the dominant nuclear pool of Bcl-2 remained stable during apoptosis, the level of the smaller mitochondrial pool was rapidly downregulated. Signals induced by anti-apoptotic cytokines continuously replenish the mitochondrial pool, but nuclear Bcl-2 is independent of such signals. Mitochondrial Bcl-2 is lost rapidly by a caspase independent mechanism in the absence of survival factors, in contrast only a small proportion of the nuclear pool of Bcl-2 is lost during the execution phase and this loss is a caspase dependent process. We conclude that these two intracellular pools of Bcl-2 are regulated through different mechanisms and only the cytokine-mediated regulation of the mitochondrial pool is relevant to the control of the initiation of apoptosis. D. Scheel-Toellner and K. Raza have contributed equally to this study.     

Upregulation of Beclin-1 expression and phosphorylation of Bcl-2 and p53 are involved in the JNK-mediated autophagic cell death

Though the activation of c-Jun NH2-terminal kinase (JNK) has been reported to be essential for autophagic cell death in response to various stressors, the molecular links between JNK activation and autophagic cell death signaling remain elusive. Here we report that, in the JNK-dependent autophagic cell death of HCT116 cells induced by an agonistic single chain variable fragment antibody, HW1, against human death receptor 5 (DR5), JNK activation upregulated Beclin-1 expression and induced Bcl-2 and p53 phosphorylation. Further, the p53-deficient HCT116 cells showed less susceptibility to the HW1-mediated autophagic cell death than the wild type cells, suggesting that JNK-mediated p53 phosphorylation promotes the autophagic cell death. Our results suggest that DR5-stimulated JNK activation and its consequent fluxes into the pro-autophagic signaling pathways contribute to the autophagic cell death in cancer cells.     

Design,synthesis and pharmacological evaluation of new acyl sulfonamides as potent and selective Bcl-2 inhibitors

The antiapoptotic protein Bcl-2, overexpressed in many tumor cells, is an attractive target for potential small molecule anticancer drug discovery. Herein, we report a different structural modification approach on ABT-263 by merging the piperazinyl-phenyl fragment into a bicyclic framework leading to a series of novel analogues, among which tetrahydroisoquinoline 13 was nearly equally potent against Bcl-2 as ABT-263. Further SAR in the P4-interaction pocket affored the difluoroazetidine substituted analogue 55, which retained good Bcl-2 activity with improved Bcl-2/Bcl-xL selectivity.     

HAMLET triggers apoptosis but tumor cell death is independent of caspases, Bcl-2 and p53

HAMLET (Human α-lactalbumin Made Lethal to Tumor cells) triggers selective tumor cell death in vitro and limits tumor progression in vivo. Dying cells show features of apoptosis but it is not clear if the apoptotic response explains tumor cell death. This study examined the contribution of apoptosis to cell death in response to HAMLET. Apoptotic changes like caspase activation, phosphatidyl serine externalization, chromatin condensation were detected in HAMLET-treated tumor cells, but caspase inhibition or Bcl-2 over-expression did not prolong cell survival and the caspase response was Bcl-2 independent. HAMLET translocates to the nuclei and binds directly to chromatin, but the death response was unrelated to the p53 status of the tumor cells. p53 deletions or gain of function mutations did not influence the HAMLET sensitivity of tumor cells. Chromatin condensation was partly caspase dependent, but apoptosis-like marginalization of chromatin was also observed. The results show that tumor cell death in response to HAMLET is independent of caspases, p53 and Bcl-2 even though HAMLET activates an apoptotic response. The use of other cell death pathways allows HAMLET to successfully circumvent fundamental anti-apoptotic strategies that are present in many tumor cells.     

Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.     

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.     

Bcl-2家族蛋白及其在细胞凋亡中的作用

Bcl-2家族蛋白是目前已知的细胞凋亡中最重要的调控因子,在细胞凋亡通路中起着重要的调节作用．对其作用机制的研究将有助于对肿瘤,自身免疫性和神经变性等疾病的治疗。该文介绍Bcl-2家族中主要的几种蛋白在凋亡中的作用,以及对线粒体膜通透性的调控作用。     

Full-length expanded ataxin-3 enhances mitochondrial-mediated cell death and decreases Bcl-2 expression in human neuroblastoma cells

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. An unstable CAG trinucleotide repeat expansion in MJD gene on long arm of chromosome 14 has been identified as the pathologic mutation of MJD and apoptosis was previously shown to be responsible for the neuronal cell death of the disease. In this study, we utilized human neuronal SK-N-SH cells stably transfected with HA-tagged full-length MJD with 78 polyglutamine repeats to examine the effects of polyglutamine expansion on neuronal cell survival in the early stage of disease. Various pro-apoptotic agents were used to assess the tolerance of the mutant cells and to compare the differences between cells with and without mutant ataxin-3. Concentration- and time-dependent experiments showed that the increase in staurosporine-induced cell death was more pronounced and accelerated in cells containing expanded ataxin-3 via MTS assays. Interestingly, under basal conditions, Western blot and immunocytochemical analyses showed a significant decrease of Bcl-2 protein expression and an increase of cytochrome c in cells containing expanded ataxin-3 when compared with those of the parental cells. The same reduction of Bcl-2 was further confirmed in fibroblast cells with mutant ataxin-3. In addition, exogenous expression of Bcl-2 desensitized SK-N-SH-MJD78 cells to poly-Q toxicity. These results indicated that mitochondrial-mediated cell death plays a role in the pathogenesis of MJD. In our cellular model, full-length expanded ataxin-3 that leads to neurodegenerative disorders significantly impaired the expression of Bcl-2 protein, which may be, at least in part, responsible for the weak tolerance to polyglutamine toxicity at the early stage of disease and ultimately resulted in an increase of stress-induced cell death upon apoptotic stress.     

Bcl-2蛋白的研究进展

Bcl-2基因属于Bcl-2基因家族,其表达的Bcl-2蛋白在细胞凋亡过程中通过抑制线粒体中细胞色素C的释放,可明显地抑制细胞凋亡。由于在大规模的细胞培养过程中,细胞的死亡以凋亡为主,所以可通过建立稳定过表达Bcl-2蛋白的重组细胞株,以抑制细胞的凋亡。同时,Bcl-2蛋白的变化不但可预见肿瘤的发生,而且通过药物降低Bcl-2的含量对于肿瘤的治疗也具有积极的意义。本文综述了Bcl-2蛋白的结构、功能、抗凋亡作用的机理,以及目前在生物制药和临床方面的应用和前景。     

Lovastatin-induced up-regulation of the BH3-only protein, Bim, and cell death in glioblastoma cells

The mechanism of lovastatin-induced cell death was examined in three established human glioblastoma cell lines; U87, U251, and U138. Changes in potential modifiers of apoptosis, including Bcl-2 family proteins and MAP kinase targets after such lovastatin treatment, were evaluated. Lovastatin (5 microm) treatment causes extensive cell death in two of the cell lines, U87 and U251; but only minimal in a third, U138. Lovastatin-induced death occurs in correlation with significantly increased levels of the BH3-only protein, Bim. The up-regulation of Bim levels was directly associated with an increased incidence of apoptosis. Lovastatin treatment in U87 cells results in activation of targets of three major mitogen-activating protein kinase cascades including Erk1/2, JNK and p38. Changes in levels of Bim, as well as increase phosphorylation of Erk1/2, c-jun, and p38 are all prevented by co-incubation of lovastatin and the isoprenylation metabolite, geranylgeranyl pyrophosphate. Inhibition of the MAP kinase pathways failed to block the increased expression of Bim expression or cell death. Further elucidation of the mechanisms of lovastatin-induced up-regulation of Bim and apoptosis in glioblastoma cells are important in determining a potential role for lovastatin as a chemotherapy agent.     

Voltage-dependent anion channels: their roles in plant defense and cell death

The voltage-dependent anion channels (VDACs), mitochondrial outer membrane components, are present in organisms from fungi to animals and plants. They are thought to function in the regulation of metabolite transport between mitochondria and the cytoplasm. Sufficient knowledge on plant VDACs has been accumulated, so that we can here summarize the current information. Then, the involvement of mitochondria in plant defense and cell death is overviewed. While, in mammals, it is suggested that VDAC, also known as a component of the permeability transition pore (PTP) complex formed in the junction site of mitochondrial outer and inner membrane, is a key player in mitochondria-mediated cell death, little is known about the role of plant VDACs in this process. We have shown that plant VDACs are involved in mitochondria-mediated cell death and in defense against a non-host pathogen. In light of the current findings, we discuss the role of the PTP complex and VDAC as its component in plant pathogen defense and cell death.