Disruption of mitochondrial quality control genes promotes caspase-resistant cell survival following apoptotic stimuli

In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases, a subpopulation of treated cells can exhibit a sublethal response, termed “minority MOMP.” In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease caspase-activated DNase (DNA fragmentation factor subunit beta). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.     

Alpha-tocopherol-mediated caspase-3 up-regulation enhances susceptibility to apoptotic stimuli

Although alpha-tocopherol is known as an essential micronutrient involved in various oxidative stress-related processes, its non-antioxidant activities have only been characterized in recent years. In this study, we reveal that (+)-alpha-tocopherol [RRR-alpha-tocopherol] enhances cellular susceptibility to both oxidative and non-oxidative apoptosis-inducing stimuli through up-regulation of caspase-3/CPP32 expression in several human cell lines. Exposure of (+)-alpha-tocopherol pretreated cells to known apoptosis-inducing stimuli, such as Fas, H(2)O(2), or etoposide, resulted in an increase in cellular apoptotsis. In addition, (+)-alpha-tocopherol also elevated the pro-caspase-3 protein level and mRNA expression in a time- and dose-dependent manner, while other tocopherol analogues showed no effect. Experiments using a GC-specific DNA binding agent, mithramycin A, and an electrophoretic mobility shift assay demonstrated that Sp1 might mediate the enhanced expression of caspase-3. Our results also confirmed that (+)-alpha-tocopherol promotes the expression, but not the activation, of caspase-3 in various human cell lines. These findings provide biological evidence showing that (+)-alpha-tocopherol can amplify the apoptotic response by up-regulating the expression of pro-caspase-3.     

Hypoxic enlarged mitochondria protect cancer cells from apoptotic stimuli

It is well established that cells exposed to the limiting oxygen microenvironment (hypoxia) of tumors acquire resistance to chemotherapy, through mechanisms not fully understood. We noted that a large number of cell lines showed protection from apoptotic stimuli, staurosporine, or etoposide, when exposed to long‐term hypoxia (72 h). In addition, these cells had unusual enlarged mitochondria that were induced in a HIF‐1‐dependent manner. Enlarged mitochondria were functional as they conserved their transmembrane potential and ATP production. Here we reveal that mitochondria of hypoxia‐induced chemotherapy‐resistant cells undergo a HIF‐1‐dependent and mitofusin‐1‐mediated change in morphology from a tubular network to an enlarged phenotype. An imbalance in mitochondrial fusion/fission occurs since silencing of not only the mitochondrial fusion protein mitofusin 1 but also BNIP3 and BNIP3L, two mitochondrial HIF‐targeted genes, reestablished a tubular morphology. Hypoxic cells were insensitive to staurosporine‐ and etoposide‐induced cell death, but the silencing of mitofusin, BNIP3, and BNIP3L restored sensitivity. Our results demonstrate that some cancer cells have developed yet another way to evade apoptosis in hypoxia, by inducing mitochondrial fusion and targeting BNIP3 and BNIP3L to mitochondrial membranes, thereby giving these cells a selective growth advantage. J. Cell. Physiol. 222: 648–657, 2010. © 2009 Wiley‐Liss, Inc.     

Dopamine- and zinc-induced autophagosome formation facilitates PC12 cell survival

Dopamine oxidation and divalent cations have been reported to induce neuronal cell death. Although autophagy is involved in neuronal cell death, it has also been suggested to facilitate cell survival. We sought to investigate the role of autophagy in PC12 cells and cultured neurons treated with dopamine and Zn²⁺. Cells expressing EGFP-LC3 were treated with high concentrations of dopamine and Zn²⁺, and the formation of EGFP-LC3 fluorescence aggregates was monitored. Our results showed a significant increase in the number of fluorescent puncta in the cytosol of PC12 cells treated with these chemicals. These treatments enhanced LC3 lipidation levels in PC12 cells. Decreasing the ATG7 protein level using specific small interference RNA (siRNA) and pretreating with phosphatidylinositol 3-phosphate kinase blockers, wortmannin and LY294002, inhibited puncta formation. Dopamine or Zn²⁺ treatment significantly elevated the intracellular Zn²⁺ concentration ([Zn²⁺] _i ); however, inhibiting the [Zn²⁺] _i elevation in dopamine-treated cells suppressed the puncta formation. LY294002 or siRNA-directed members of the autophagy pathway increased the fraction of phosphatidylserine present on the outer membrane leaflet in PC12 cells treated with dopamine or Zn²⁺, suggesting an increase in apoptosis. Primary embryonic midbrain neurons expressing EGFP-LC3 also displayed a significant increase in the number of fluorescent aggregates in cells upon treatment with dopamine or Zn²⁺. Dopamine or Zn²⁺ treatment significantly elevated the [Zn²⁺] _i in neurons and caused neuronal death. Our results indicate that treating cells with dopamine and Zn²⁺ results in the activation of the autophagy pathway in an effort to enhance cell survival.     

Increased chromatin accessibility facilitates intron retention in specific cell differentiation states

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including several cancers. Despite consistent reports demonstrating that intrinsic sequence features can help introns evade splicing, conflicting findings about cell type- or condition-specific IR regulation by trans-regulatory and epigenetic mechanisms demand an unbiased and systematic analysis of IR in a controlled experimental setting. We integrated matched mRNA sequencing (mRNA-Seq), whole-genome bisulfite sequencing (WGBS), nucleosome occupancy methylome sequencing (NOMe-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) data from primary human myeloid and lymphoid cells. Using these multi-omics data and machine learning, we trained two complementary models to determine the role of epigenetic factors in the regulation of IR in cells of the innate immune system. We show that increased chromatin accessibility, as revealed by nucleosome-free regions, contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. This study suggests an important role for chromatin architecture in IR regulation. With an increasing appreciation that pathogenic alterations are linked to RNA processing, our findings may provide useful insights for the development of novel therapeutic approaches that target aberrant splicing.     

Balance of apoptotic cell death and survival in allergic diseases

Allergic diseases result from over-reaction of the immune system in response to exogenous allergens, where inflammatory cells have constantly extended longevity and contribute to an on-going immune response in allergic tissues. Here, we review disequilibrium in the death and survival of epithelial cells and inflammatory cells in the pathological processes of asthma, atopic dermatitis, and other allergic diseases.     

Lysophosphatidic acid as a novel cell survival/apoptotic factor

Lysophosphatidic acid (LPA) activates its cognate G protein-coupled receptors (GPCRs) LPA(1-3) to exert diverse cellular effects, including cell survival and apoptosis. The potent survival effect of LPA on Schwann cells (SCs) is mediated through the pertussis toxin (PTX)-sensitive G(i/o)/phosphoinositide 3-kinase (PI3K)/Akt signaling pathways and possibly enhanced by the activation of PTX-insensitive Rho-dependent pathways. LPA promotes survival of many other cell types mainly through PTX-sensitive G(i/o) proteins. Paradoxically, LPA also induces apoptosis in certain cells, such as myeloid progenitor cells, hippocampal neurons, and PC12 cells, in which the activation of the Rho-dependent pathways and caspase cascades has been implicated. The effects of LPA on both cell survival and apoptosis underscore important roles for this lipid in normal development and pathological processes.     

Production and characterization of a monoclonal antibody able to discriminate galectin-1 from galectin-2 and galectin-3

Antisera raised against galectin-1 exhibit crossreactivities with other galectins or related molecules. In order to overcome this problem, a monoclonal antibody to human brain galectin-1 was obtained by selecting clones without reactivity toward galectin-3. This mAb specifically bound galectin-1 of various animal origins but neither galectin-2 nor galectin-3. Western-blotting analysis of soluble human brain extracts after 2D gel electrophoresis revealed only the two most acidic isoforms of galectin-1. The ability of this mAb to bind galectin-1/asialofetuin complexes indicates that its epitope is not localized in the carbohydrate recognition domain of galectin-1. This particularity induces with efficiency its monospecificity.      

Sialylation of beta1 integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis

In previous studies, we determined that beta1 integrins from human colon tumors have elevated levels of alpha2-6 sialylation, a modification added by beta-galactosamide alpha-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the beta1 integrin is thought to be a ligand for galectin-3 (gal-3), a tumor-associated lectin. The effects of gal-3 are complex; intracellular forms typically protect cells against apoptosis through carbohydrate-independent mechanisms, whereas secreted forms bind to cell surface oligosaccharides and induce apoptosis. In the current study, we tested whether alpha2-6 sialylation of the beta1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking alpha2-6 sialylation exhibit beta1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of alpha2-6 sialic acids from ST6Gal-I expressors by neuraminidase treatment restores gal-3 binding. Additionally, using a blot overlay approach, we determined that gal-3 binds directly and preferentially to unsialylated, as compared with alpha2-6-sialylated, beta1 integrins. To understand the physiologic consequences of gal-3 binding, cells were treated with gal-3 and monitored for apoptosis. Galectin-3 was found to induce apoptosis in parental SW48 colonocytes (unsialylated), whereas ST6Gal-I expressors were protected. Importantly, gal-3-induced apoptosis was inhibited by function blocking antibodies against the beta1 subunit, suggesting that beta1 integrins are critical transducers of gal-3-mediated effects on cell survival. Collectively, our results suggest that the coordinate up-regulation of gal-3 and ST6Gal-I, a feature that is characteristic of colon carcinoma, may confer tumor cells with a selective advantage by providing a mechanism for blockade of the pro-apoptotic effects of secreted gal-3.     

Serum/glucocorticoid-induced protein kinase-1 facilitates androgen receptor-dependent cell survival

Androgen receptor (AR) is a critical factor in the development and progression of prostate cancer. We and others recently demonstrated that eliminating AR expression leads to apoptotic cell death in AR-positive prostate cancer cells. To understand the mechanisms of AR-dependent survival, we performed a genome-wide search for AR-regulated survival genes. We found that serum/glucocorticoid-induced protein kinase-1 (SGK-1) mRNA levels were significantly upregulated after androgen stimulation, which was confirmed to be AR dependent. Promoter analysis revealed that the AR interacted with the proximal and distal regions of the sgk1 promoter, leading to sgk-1 promoter activation after androgen stimulation. Functional assays demonstrated that SGK-1 was indispensable for the protective effect of androgens on cell death induced by serum starvation. SGK-1 overexpression not only rescued cells from AR small-interfering RNA (siRNA)-induced apoptosis, but also enhanced AR transactivation, even in the absence of androgen. Additionally, SGK-1 siRNA reduced AR transactivation, indicating a positive feedback effect of SGK-1 expression on AR-mediated gene expression and cellular survival. Taken together, our data suggest that SGK-1 is an androgen-regulated gene that plays a pivotal role in AR-dependent survival and gene expression.     

Role of BAG3 protein in leukemia cell survival and response to therapy

The ability of BAG3, a member of the BAG family of heat shock protein (Hsp) 70 - cochaperones, to sustain the survival of human primary B-CLL and ALL cells was recognized about nine years ago. Since then, the anti-apoptotic activity of BAG3 has been confirmed in other tumor types, where it has been shown to regulate the intracellular concentration and localization of apoptosis-regulating factors, including NF-κB-activating (IKKγ) and Bcl2-family (Bax) proteins. Furthermore, growing evidences support its role in lymphoid and myeloid leukemia response to therapy. Moreover in the last years, the contribution of BAG3 to autophagy, a process known to be involved in the pathogenesis and response to therapy of leukemia cells, has been disclosed, opening a new avenue for the interpretation of the role of this protein in leukemias' biology.     

Cytotoxic and apoptotic effects of boron compounds on leukemia cell line

In this study we investigated the effects of boric acid and sodium tetraborate on an acute leukemia cell line and healthy human lymphocytes. We evaluated the effects of boric acid and sodium tetraborate on the HL-60 cell line and healthy human lymphocytes by using the methods of MTT, Neutral Red, AO (flow cytometry) and transmission electron microscope. We found that there were dead cells at a concentration of 500 µM boric acid and sodium tetraborate (50 % and 40 %, respectively). An apoptotic effect was found at a concentration of 1,000 µM concentration in normal lymphocytes and HL-60 (acute leukemia cells) cells (2.5 % and 8.8 % respectively). We observed that boric acid at a concentration of 500 µM caused double nucleus and micronucleus formation in both HL-60 cells and lymphocytes. An expansion in mitochondrial dimension and deformation in cristas also appeared. Our findings suggest that boric acid is more effective than sodium tetraborate on the HL-60, and boric acid in particular showed a cytotoxic effect on HL-60 in comparison to healthy lymphocytes and it also affected the mitochondrial pathway.     

The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8

Apoptosis resistance is a hurdle for cancer treatment. HECTD3, a new E3 ubiquitin ligase, interacts with caspase-8 death effector domains and ubiquitinates caspase-8 with K63-linked polyubiquitin chains that do not target caspase-8 for degradation but decrease the caspase-8 activation. HECTD3 depletion can sensitize cancer cells to extrinsic apoptotic stimuli. In addition, HECTD3 inhibits TNF-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 cleavage in an E3 ligase activity-dependent manner. Mutation of the caspase-8 ubiquitination site at K215 abolishes the HECTD3 protection from TRAIL-induced cleavage. Finally, HECTD3 is frequently overexpressed in breast carcinomas. These findings suggest that caspase-8 ubiquitination by HECTD3 confers cancer cell survival.     

Multifaceted role of galectin-3 on human glioblastoma cell motility

Astrocytic tumors' aggressiveness results from an imbalance between cell proliferation and cell death favoring growth, but also from the propensity of tumor cells to detach from the primary tumor site, migrate, and invade the surrounding parenchyma. Astrocytic tumor progression is known to be associated with an increased expression of galectin-3. We investigated in cell culture how galectin-3 expression affects astrocytoma cell motility. Galectin-3 deficient cells were obtained by stable transfection of the U373 glioblastoma cell line with a specific expression antisense plasmid. Cultured galectin-3 deficient glioblastoma cells showed increased motility potential on laminin and modifications in the cytoskeleton reorganization. In addition, c-DNA microarrays and quantitative immunofluorescence analysis showed that galectin-3 deficient U373 cells have an increased expression of integrins-alpha6 and -beta1, proteins known to be implicated in the regulation of cell adhesion.     

Caspase-3 activation is an early event and initiates apoptotic damage in a human leukemia cell line

Many apoptotic pathways culminate in the activation of caspase cascades usually triggered by the apical caspases-8 or -9. We describe a paradigm where apoptosis is initiated by the effector caspase-3. Diethylmaleate (DEM)-induced apoptotic damage in Jurkat cells was blocked by the anti-apoptotic protein Bcl-2, whereas, a peptide inhibitor of caspase-3 but not caspase-9 blocked DEM-induced mitochondrial damage. Isogenic Jurkat cell lines deficient for caspase-8 or the adaptor FADD (Fas associated death domain) were not protected from DEM-induced apoptosis. Caspase-3 activation preceded that of caspase-9 and initial processing of caspase-3 was regulated independent of caspase-9 and Bcl-2. However, inhibitors of caspase-9 or caspase-6 regulated caspase-3 later in the pathway. We explored the mechanism by which caspase-3 processing is regulated in this system. DEM triggered a loss of Erk-1/2 phosphorylation and XIAP (X-linked inhibitor of apoptosis protein) expression. The phorbol ester PMA activated a MEK-dependent pathway to block caspase-3 processing and cell death. Constitutively active MEK-1 (CA-MEK) upregulated XIAP expression and exogenous XIAP inhibited DEM-induced apoptotic damage. Thus, we describe a pathway where caspase-3 functions to initiate apoptotic damage and caspase-9 and caspase-6 amplify the apoptotic cascade. Further, we show that MEK may regulate caspase-3 activation via the regulation of XIAP expression in these cells.     

The Tim-3 ligand galectin-9 negatively regulates CD8+ alloreactive T cell and prolongs survival of skin graft

CD8+ alloreactive T cells are the key mediators of accelerated rejection. Vigorous CD8+ alloreactive T cells responses against alloantigens, which is the main effector mechanism in acute allograft rejection, has been well described. But the molecular mechanisms to dampen activated CD8+ T cells are largely unknown. On the other hand, Tim-3 is a molecule expressed on terminally differentiated CD4+ Th1 cells. Engaging Tim-3 with its ligand galectin-9 causes an inhibitory signal, resulting in apoptosis of Th1 cells and negatively regulates Th1 type immunity. However, the question whether CD8+ T cells express surface molecular Tim-3 has not been fully elucidated. In this study, we have investigated which CD8+ subset express molecular Tim-3 by flow cytometric assay. In addition, cytotoxic assay was applied to analyze whether CD8+ alloreactive T cells were sensitive to galectin-9 induced apoptosis. Here, our results demonstrated that Tim-3 was expressed on activated CD8+ alloreactive T cells (CD8+CD44highCD62Llow), but not expressed on na?ve CD8+ T cells. Furthermore, alloreactive CD8+ cytotoxic T cells were sensitive to galectin-9 induced apoptosis both in vitro and vivo, resulting in attenuation of CD8+ alloreactive T cells mediated cytotoxicity and prolonged survival of skin graft.     

Armepavine oxalate induces cell death on CCRF-CEM leukemia cell line through an apoptotic pathway

Drug-induced cell death can occur as a result of DNA damage, which in turn may lead to the reduction of bcl-2 expression and activation of caspase-3 expression. In the present study, we investigated the effect of armepavines and atherosperminine on the cell survival rate and expression of bcl-2 and caspase-3 in CCRF-CEM cells. Our data have revealed that armepavine oxalate reduced the survival rate of CCRF-CEM cells in a dose- and time-dependent manner by MTT assay. However, no significant effects of armepavine MeI and atherosperminine N-oxide on the survival rate of the CCRF-CEM cell were observed. Armepavine oxalate-induced cell death was considered to be apoptotic on the basis of observed formation of the DNA ladder and the typical apoptotic morphological change by Hoechst 33258 staining. The expression of bcl-2 protein in CCRF-CEM cells treated with 30 microM armepavine oxalate was significantly decreased in western blotting analysis. In contrast, the expression of active caspase-3 in the cells was increased by armepavine oxalate in a dose-dependent manner. These findings indicate the involvement of bcl-2 and caspase-3 in the apoptotic process of CCRF-CEM cells induced by armepavine oxalate. The increased expression of active caspase-3 as well as decreased expression of bcl-2 support the assumption the armepavine oxalate-treated cells may be capable to complete the entire apoptotic process ending in cell fragmentation.     

lncRNA SNHG3 facilitates acute myeloid leukemia cell growth via the regulation of miR-758-3p/SRGN axis

Small nucleolar RNA host gene 3 (SNHG3) is a newly identified long non-coding RNA whose dysregulation has been reported in several cancers. However, the details about clinical significances and biological functions of SNHG3 on acute myeloid leukemia (AML) remain covered. In this study, we revealed increased SNHG3 expression in AML samples and cells and its high potential as a prognostic biomarker for AML patients. Likewise, serglycin (SRGN), which plays an important role in granule-mediated apoptosis, was previously verified to be upregulated in AML and confirmed again by the present study, and its upregulation predicted poor outcomes in AML. Furthermore, knockdown of SNHG3 or SRGN inhibited cell proliferation and induced cell apoptosis. Besides, silencing SNHG3 noticeably decreased the expression of SRGN in AML cells. Moreover, we uncovered that SNHG3 modulated SRGN expression by competitively binding with miR-758-3p. Importantly, both miR-758-3p suppression and SRGN overexpression could mitigate the inhibitory effects of SNHG3 depletion on AML cell growth. Intriguingly, the higher SRGN expression in AML samples with a higher SNHG3 level exhibited an enhanced Ki67 level but a reduced caspase 3 level. To sum up, SNHG3 elicits a growth-promoting function in AML via sponging miR-758-3p to regulate SRGN expression, providing a new therapeutic road for AML patients.