Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways

Survivin inhibits apoptosis during development and carcinogenesis and is absent in differentiated cells. To determine whether survivin inhibition induces cell death in neural tumor cells, survivin antisense oligonucleotides (SAO) were administered to a human neuroblastoma (MSN) and an oligodendroglioma (TC620) resulting in a dose-dependent reduction in survivin protein. Although 74% of the SAO-treated MSN cells were trypan blue(+), PARP cleavage or activated caspase-3 was not observed. However nuclear translocation of AIF occurred and XIAP increased dramatically. Co-administration of z-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk) with SAO did not inhibit cell death suggesting a caspase-independent mechanism of cell death. Propidium iodide (PI) staining revealed multiple large macronuclei with no apoptotic bodies supporting a role for survivin in cell division. By contrast, while 70% of the SAO-treated TC620 cells were trypan blue(+), PARP was cleaved, cells were TUNEL(+) and PI-staining revealed macronuclei and numerous apoptotic bodies. Co-treatment of the TC620 cells with SAO and zVAD-fmk blocked cell death. While no macronuclei or apoptotic bodies were observed there was a two-fold increase in metaphase cells. Our results suggest that survivin inhibition decreases the viability of human neural tumor cells and as a result of mitotic catastrophe, cell death can be initiated by either a classic apoptotic mechanism or a caspase-independent mechanism.     

Role of the JNK/c-Jun/AP-1 signaling pathway in galectin-1-induced T-cell death

Galectin-1 (gal-1), an endogenous β-galactoside-binding protein, triggers T-cell death through several mechanisms including the death receptor and the mitochondrial apoptotic pathway. In this study we first show that gal-1 initiates the activation of c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 4 (MKK4), and MKK7 as upstream JNK activators in Jurkat T cells. Inhibition of JNK activation with sphingomyelinase inhibitors (20 μM desipramine, 20 μM imipramine), with the protein kinase C-δ (PKCδ) inhibitor rottlerin (10 μM), and with the specific PKCθ pseudosubstrate inhibitor (30 μM) indicates that ceramide and phosphorylation by PKCδ and PKCθ mediate gal-1-induced JNK activation. Downstream of JNK, we observed increased phosphorylation of c-Jun, enhanced activating protein-1 (AP-1) luciferase reporter, and AP-1/DNA-binding in response to gal-1. The pivotal role of the JNK/c-Jun/AP-1 pathway for gal-1-induced apoptosis was documented by reduction of DNA fragmentation after inhibition JNK by SP600125 (20 μM) or inhibition of AP-1 activation by curcumin (2 μM). Gal-1 failed to induce AP-1 activation and DNA fragmentation in CD3-deficient Jurkat 31-13 cells. In Jurkat E6.1 cells gal-1 induced a proapoptotic signal pattern as indicated by decreased antiapoptotic Bcl-2 expression, induction of proapoptotic Bad, and increased Bcl-2 phosphorylation. The results provide evidence that the JNK/c-Jun/AP-1 pathway plays a key role for T-cell death regulation in response to gal-1 stimulation.     

Induction of programmed cell death in human retinoblastoma Y79 cells by C2-ceramide

C₂-ceramide, a cell-permeable analogue of ceramide, induced significant, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characterized by microscopic evidence of cell shrinkage, membrane blebbing, nuclear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexin V-FITC. Apoptosis was also assessed by monitoring changes in cell granularity by staining with the combined fluorescent dyes acridine orange and ethidium bromide. C₂-ceramide induced these morphological changes without a concomitant production of oligonucleosomal fragments responsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C₂-ceramide, as C₂-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydrolysis were ineffective. The effect also depended on mRNA and protein synthesis as it was markedly inhibited by actinomycin D and cycloheximide. Sphingomyelinase and interleukin-l, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induced apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.     

Activation—induced cell death in B lymphocytes

Upon encountering the antigen(Ag),the immune system can either develop a specific immune response of enter a specific state of unresponsiveness,tolerance.The response of B cells to their specific Ag can be activation and proliferation,leading to the immune response,or anergy and activation-induced cell death(AICD),leading to tolerance.AICD in B lymphocytes is a highly regulated event initiated by crosslinking of the B cell receptor (BCR).BCR engagement initiates several signaling events such as activation of PLCγ,Ras,and PI3K,which generally speaking,lead to survival.However,in the absence of survival signals(CD40 or IL-4R engagement),BCR crosslinking can also promote apoptotic signal transduction pathways such as activation of effector caspases,expression of pro-apoptotic genes,and inhibition of pro-survival genes.The complex interplay between survival and death signals determines the B cell fate and, consequently,the immune response.     

A role for the actin cytoskeleton in cell death and aging in yeast

Several determinants of aging, including metabolic capacity and genetic stability, are recognized in both yeast and humans. However, many aspects of the pathways leading to cell death remain to be elucidated. Here we report a role for the actin cytoskeleton both in cell death and in promoting longevity. We have analyzed yeast strains expressing mutants with either increased or decreased actin dynamics. We show that decreased actin dynamics causes depolarization of the mitochondrial membrane and an increase in reactive oxygen species (ROS) production, resulting in cell death. Important, however, is the demonstration that increasing actin dynamics, either by a specific actin allele or by deletion of a gene encoding the actin-bundling protein Scp1p, can increase lifespan by over 65%. Increased longevity appears to be due to these cells producing lower than wild-type levels of ROS. Homology between Scp1p and mammalian SM22/transgelin, which itself has been isolated in senescence screens, suggests a conserved mechanism linking aging to actin stability.     

Rotenone-induced caspase 9/3-independent and -dependent cell death in undifferentiated and differentiated human neural stem cells

We used human neural stem cells (hNSCs) and their differentiated cultures as a model system to evaluate the mechanism(s) involved in rotenone (RO)- and camptothecin (CA)-induced cytotoxicity. Results from ultrastructural damage and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining indicated that RO-induced cytotoxicity resembled CA-induced apoptosis more than H(2)O(2)-induced necrosis. However, unlike CA-induced, caspase 9/3-dependent apoptosis, there was no increased activity in caspase 9, caspase 3 or poly (ADP-ribose) polymerase (PARP) cleavage in RO-induced cytotoxicity, in spite of time-dependent release of cytochrome c and apoptosis-inducing factor (AIF) following mitochondrial membrane depolarization and a significant increase in reactive oxygen species generation. Equal doses of RO and CA used in hNSCs induced caspase 9/3-dependent apoptosis in differentiated cultures. Time-dependent ATP depletion occurred earlier and to a greater extent in RO-treated hNSCs than in CA-treated hNSCs, or differentiated cultures treated with RO or CA. In conclusion, these results represent a unique ultrastructural and molecular characterization of RO- and CA-induced cytotoxicity in hNSCs and their differentiated cultures. Intracellular ATP levels may play an important role in determining whether neural progenitors or their differentiated cells follow a caspase 9/3-dependent or -independent pathway in response to acute insults from neuronal toxicants.     

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.     

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.     

Immortalization of human T lymphocytes by oncogenes

Immortalized human T cell lines were established by cotransfecting c-Ha-ras and c-myc oncogenes to lymph node lymphocytes. The cell lines kept growing for 3 months after establishment without a decrease in growth rate. The cells did not require interleukin-2(IL-2) for their growth, but addition of IL-2 stimulated the growth of these cells. Flow cytometric analysis revealed that these cells were T cells expressing CD4 or CD8 antigens. A CD4 positive (CD4⁺) cell line produced IL-6, indicating that the cell line belongs to helper T cells. The CD8 positive (CD8⁺) cell line possessed cytotoxicity to tumor cells, indicating that the cell line were killer T cells. Both cell lines were able to proliferate in serum-free medium indefinitely.     

Protective role of HSP27 against UVC-induced cell death in human cells

It is an intriguing problem whether heat shock proteins (HSPs) play a protective role in UVC-induced cell death in human cells, and the problem has not been solved. To search for the HSPs involved in UVC resistance, gene expression profiles using cDNA array were compared between UVC-sensitive human RSa cells and their UVC-resistant variant AP(r)-1 cells. The expression levels of heat shock protein 27 (HSP27) were lower in RSa cells than in AP(r)-1 cells. RSa cells transfected with sense HSP27 cDNA showed slightly lower sensitivity to UVC-induced cell death than the control cells transfected with a vector alone and much lower sensitivity than RSa cells transfected with the antisense HSP27 cDNA. Furthermore, the removal capacities of the two major types of UVC-damaged DNA (thymine dimers and (6-4)photoproducts) in the cells with the up-regulation of HSP27 were moderately elevated compared with those in the control cells, while those in the cells with down-regulation were remarkably suppressed. These results suggest that HSP27 is involved in the UVC-resistance of human cells, at least those tested, possibly via functioning in nucleotide excision repair.     

Familial NK cell deficiency associated with impaired IL-2- and IL-15-dependent survival of lymphocytes

We previously reported the clinical phenotype of two siblings with a novel inherited developmental and immunodeficiency syndrome consisting of severe intrauterine growth retardation and the impaired development of specific lymphoid lineages, including transient CD8 alphabeta T lymphopenia and a persistent lack of blood NK cells. We describe here the elucidation of a plausible underlying pathogenic mechanism, with a cellular phenotype of impaired survival of both fresh and herpesvirus saimiri-transformed T cells, in the surviving child. Clearly, NK cells could not be studied. However, peripheral blood T lymphocytes displayed excessive apoptosis ex vivo. Moreover, the survival rates of CD4 and CD8 alphabeta T cell blasts generated in vitro, and herpesvirus saimiri-transformed T cells cultured in vitro, were low, but not nil, following treatment with IL-2 and IL-15. In contrast, Fas-mediated activation-induced cell death was not enhanced, indicating a selective excess of cytokine deprivation-mediated apoptosis. In keeping with the known roles of IL-2 and IL-15 in the development of NK and CD8 T cells in the mouse model, these data suggest that an impaired, but not abolished, survival response to IL-2 and IL-15 accounts for the persistent lack of NK cells and the transient CD8 alphabeta T lymphopenia documented in vivo. Impaired cytokine-mediated lymphocyte survival is likely to be the pathogenic mechanism underlying this novel form of inherited and selective NK deficiency in humans.     

IL-2 protects T lymphocytes from glucocorticoid-induced DNA fragmentation and cell death

In the IL-2-dependent T cell clone CTLL-2, dexamethasone, a synthetic glucocorticoid, induces a suicide program characterized by the early degradation of chromatin in oligonucleosome-length fragments which precedes the loss of cell viability by 2 to 4 h. These effects are most likely mediated through the interaction with a specific glucocorticoid receptor as suggested by the structure-activity relationship of the various steroids tested. Incubation of nuclei of glucocorticoid-untreated cells in the presence of calcium and magnesium ions induces the cleavage of DNA in the linker region between nucleosomes, suggesting that fragmentation of chromatin in intact cells by glucocorticoids may involve the activation of a preexisting endonuclease. Interestingly, the presence of a saturating dose of IL-2 during the treatment of CTLL-2 cells with glucocorticoids completely blocks the cell death program.     

Role of cell membrane Na,K-ATPase for survival of human lymphocytes in vitro

Lymphocytes are primordial immune cells with variable life times. Besides genetic programming, extracellular factors interacting with cell surface receptors might alter cell survival. We investigated whether the activity of the membrane-embedded Na,K-ATPase (EC 3.6.1.37) or sodium pump (NKA) plays a role for cell survival since this ubiquitous system establishes the vital transmembrane Na and K gradients as well as the resulting high intracellular K/Na ratio required for macromolecule synthesis; furthermore, the system exposes an extracellular inhibitory receptors for cardioactive steroids and palytoxin. Isolated human lymphocytes were incubated in vitro and their viability assessed by exclusion of trypan blue. Various incubation conditions were compared; in RPMI-1640 medium cell viability was preserved for 30 h at 37 °C. Externally added ouabain, a hydrophilic cardioactive steroid, blocked the [⁸⁶Rb]potassium uptake at nanomolar concentrations. Despite pump inhibition ouabain did not alter lymphocyte survival, even at 10 mM for 30 h. By contrast, the hydrophilic toxin palytoxin, the most potent animal poison described so far, killed all cells within 2 h at 10 nM; this toxin is known to act via the sodium pump and to provoke deadly cation-leaks by unmasking a channel component. Intracellular Na increased and K decreased as measured by atomic absorption spectrometry in presence of palytoxin; cell swelling was seen by electron microscopy. Ouabain protected the cells from the toxic effect of palytoxin. The results reveal a pivotal role of NKA integrity for lymphocyte survival.Abbreviations BCA bicinchonic acid - D-PBS Dulbecco's Phosphate Buffered Saline - HBSS Hanks' Balanced Salt Solution - PYX palytoxin (used in figures only) - NKA Na,K-ATPase     

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.     

p53-dependent neuronal cell death in a DJ-1-deficient zebrafish model of Parkinson's disease

Mutations in DJ-1 lead to early onset Parkinson's disease (PD). The aim of this study was to elucidate further the underlying mechanisms leading to neuronal cell death in DJ-1 deficiency in vivo and determine whether the observed cell loss could be prevented pharmacologically. Inactivation of DJ-1 in zebrafish, Danio rerio, resulted in loss of dopaminergic neurons after exposure to hydrogen peroxide and the proteasome inhibitor MG132. DJ-1 knockdown by itself already resulted in increased p53 and Bax expression levels prior to toxin exposure without marked neuronal cell death, suggesting subthreshold activation of cell death pathways in DJ-1 deficiency. Proteasome inhibition led to a further increase of p53 and Bax expression with widespread neuronal cell death. Pharmacological p53 inhibition either before or during MG132 exposure in vivo prevented dopaminergic neuronal cell death in both cases. Simultaneous knockdown of DJ-1 and the negative p53 regulator mdm2 led to dopaminergic neuronal cell death even without toxin exposure, further implicating involvement of p53 in DJ-1 deficiency-mediated neuronal cell loss. Our study demonstrates the utility of zebrafish as a new animal model to study PD gene defects and suggests that modulation of downstream mechanisms, such as p53 inhibition, may be of therapeutic benefit.     

Newly synthesized AIFM1 determines the hypersensitivity of T lymphocytes to STING activation-induced cell apoptosis

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Interleukin-2-dependent regulation of Na/K pump in human lymphocytes

The present study provides the first evidence that the abundance of catalytic alpha1-subunit of Na,K-ATPase increases in the course of T cell blast transformation. Immunodepressant cyclosporin A at anti-proliferative doses diminished the induction of alpha1 protein in activated lymphocytes. Furthermore, in competent T cells, IL-2 increases both the transport activity of Na/K pump and the content of Na,K-ATPase alpha1 protein in a time-dependent manner. A correlation was found between the long-term elevation in ouabain-sensitive Rb influxes and the increase in alpha1 protein content in late activated T cells. These results suggest that (1) the increased expression of Na,K-ATPase proteins underlie the cell cycle-dependent upregulation of ion pump during T cell transformation, and (2) IL-2 is involved in the regulated expression of Na,K-ATPase in human lymphocytes.     

Effects of diethylstilbestrol in human lymphocytes in vitro: A dose and time-dependent study on genotoxic, cytotoxic and apoptotic effects

Most of the biological, chemical or physical agents that cause cell death in certain doses and time of exposure may induce either apoptosis or necrosis. This study explores in what ways the genotoxic, cytotoxic and apoptotic effects of diethylstilbestrol (DES), a chemical agent currently used in the treatment of various types of cancer, on the human lymphocytes depend upon the dose and the exposure time. For this purpose, firstly it aims to determine in what dosages and durations of DES treatment, genotoxicity and cytotoxicity in human lymphocytes occur in vitro. Secondly, it explores the effects of DES on sister-chromatid exchanges (SCEs) and apoptosis and their relation with the nitric oxide (NO) levels. Finally, it investigates whether different dosages of DES and duration of treatment with it are correlated with each other. In so doing, we investigated the relationship among the viability, necrosis and apoptosis rates of human lymphocytes which were treated with five different DES concentrations (1, 5, 10, 15 and 20 μM) for 24, 48 and 72 h, DNA fragmentation analysis of these cells, their mean SCE values and NO levels. We concluded that 5 μM DES at 24 h is the most effective dosage that induces typical features of apoptosis in human lymphocytes. Despite the fact that there are many other studies on the effects of DES on the cancer cells, we thought it might be worth looking into the effects of DES on human lymphocytes in vitro. We meant the present study to contribute to the research done in the field of cancer treatment. (Mol Cell Biochem 276: 45–53, 2005)