| 1. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 2. |
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| 3. |
- Blomgran, Robert, 1975-
(författare)
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Microbe-induced apoptosis in phagocytic cells and its role in innate immunity
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process.We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of E. coli decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis.Type 1 fimbria-expressing E. coli adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of E. coli, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production per se is important.Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.
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| 4. |
- Daré, Elisabetta, et al.
(författare)
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Methylmercury and H2O2 provoke lysosomal damage in human astrocytoma D384 cells followed by apoptosis
- 2001
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Ingår i: Free Radical Biology & Medicine. - Linköping : Elsevier. - 0891-5849 .- 1873-4596. ; 30:12, s. 1347-1356
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Tidskriftsartikel (refereegranskat)abstract
- Methylmercury (MeHg) is a neurotoxic agent acting via diverse mechanisms, including oxidative stress. MeHg also induces astrocytic dysfunction, which can contribute to neuronal damage. The cellular effects of MeHg were investigated in human astrocytoma D384 cells, with special reference to the induction of oxidative-stress-related events. Lysosomal rupture was detected after short MeHg-exposure (1 μM, 1 h) in cells maintaining plasma membrane integrity. Disruption of lysosomes was also observed after hydrogen peroxide (H2O2) exposure (100 μM, 1 h), supporting the hypothesis that lysosomal membranes represent a possible target of agents causing oxidative stress. The lysosomal alterations induced by MeHg and H2O2 preceded a decrease of the mitochondrial potential. At later time points, both toxic agents caused the appearance of cells with apoptotic morphology, chromatin condensation, and regular DNA fragmentation. However, MeHg and H2O2 stimulated divergent pathways, with caspases being activated only by H2O2. The caspase inhibitor z-VAD-fmk did not prevent DNA fragmentation induced by H2O2, suggesting that the formation of high-molecular-weight DNA fragments was caspase independent with both MeHg and H2O2. The data point to the possibility that lysosomal hydrolytic enzymes act as executor factors in D384 cell death induced by oxidative stress.
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| 5. |
- Forsberg, Jeremy, et al.
(författare)
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A caspase-2-RFXANK interaction and its implication for MHC class II expression
- 2018
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Ingår i: Cell Death and Disease. - : Springer Science and Business Media LLC. - 2041-4889 .- 2041-4889. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Despite recent achievements implicating caspase-2 in tumor suppression, the enzyme stands out from the apoptotic caspase family as a factor whose function requires further clarification. To specify enzyme characteristics through the definition of interacting proteins in apoptotic or non-apoptotic settings, a yeast 2-hybrid (Y2H) screen was performed using the full-length protein as bait. The current report describes the analysis of a captured prey and putative novel caspase-2 interacting factor, the regulatory factor X-associated ankyrin-containing protein (RFXANK), previously associated with CIITA, the transactivator regulating cell-type specificity and inducibility of MHC class II gene expression. The interaction between caspase-2 and RFXANK was verified by co-immunoprecipitations using both exogenous and endogenous proteins, where the latter approach suggested that binding of the components occurs in the cytoplasm. Cellular co-localization was confirmed by transfection of fluorescently conjugated proteins. Enhanced caspase-2 processing in RFXANK-overexpressing HEK293T cells treated with chemotherapeutic agents further supported Y2H data. Yet, no distinct differences with respect to MHC class II expression were observed in plasma membranes of antigen-presenting cells derived from wild type and caspase-2(-/-) mice. In contrast, increased levels of the total MHC class II protein was evident in protein lysates from caspase-2 RNAi-silenced leukemia cell lines and B-cells isolated from gene-targeted mice. Together, these data identify a novel caspase-2-interacting factor, RFXANK, and indicate a potential non-apoptotic role for the enzyme in the control of MHC class II gene regulation.
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| 6. |
- Gorbunova, Anna S., et al.
(författare)
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Prognostic signature based on mitochondria quality control proteins for the prediction of lung adenocarcinoma patients survival
- 2023
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Ingår i: Cell Death Discovery. - : Springer Nature. - 2058-7716. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Lung cancer is the leading cause of cancer mortality worldwide. In recent years, the incidence of lung cancer subtype lung adenocarcinoma (LUAD) has steadily increased. Mitochondria, as a pivotal site of cell bioenergetics, metabolism, cell signaling, and cell death, are often dysregulated in lung cancer cells. Mitochondria maintenance and integrity depend on mitochondrial quality control proteins (MQCPs). During lung cancer progression, the levels of MQCPs could change and promote cancer cell adaptation to the microenvironment and stresses. Here, univariate and multivariate proportional Cox regression analyses were applied to develop a signature based on the level of MQCPs (dimeric form of BNIP3, DRP1, and SIRT3) in tumorous and non-tumorous samples of 80 patients with LUAD. The MQCP signature could be used to separate the patients with LUAD into high- and low-risk groups. Survival analysis indicated that patients in the high-risk group had dramatically shorter overall survival compared with the low-risk patients. Moreover, a nomogram combining clinicopathologic features and the MQCP signature was constructed and validated to predict 1-, 3-, and 5-year overall survival of the patients. Thus, this study presents a novel signature based on MQCPs as a reliable prognostic tool to predict overall survival for patients with LUAD.
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| 7. |
- Haag, Petra, et al.
(författare)
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Caspase-2 is a mediator of apoptotic signaling in response to gemtuzumab ozogamicin in acute myeloid leukemia
- 2022
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Ingår i: Cell Death Discovery. - : Springer Nature. - 2058-7716. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- The antibody conjugate gemtuzumab ozogamicin (GO; Mylotarg((R))) provides targeted therapy of acute myeloid leukemia (AML), with recent approvals for patients with CD33-positive disease at diagnosis or relapse, as monotherapy or combined with chemotherapeutics. While its clinical efficacy is well documented, the molecular routes by which GO induces AML cell death warrant further analyses. We have earlier reported that this process is initiated via mitochondria-mediated caspase activation. Here we provide additional data, focusing on the involvement of caspase-2 in this mechanism. We show that this enzyme plays an important role in triggering apoptotic death of human AML cells after exposure to GO or its active moiety calicheamicin. Accordingly, the caspase-2 inhibitor z-VDVAD-fmk reduced GO-induced caspase-3 activation. This finding was validated with shRNA and siRNA targeting caspase-2, resulting in reduced caspase-3 activation and cleavage of poly [ADP-ribose] polymerase 1 (PARP-1). We previously demonstrated that GO-induced apoptosis included a conformational change of Bax into a pro-apoptotic state. Present data reveal that GO-treatment also induced Bid cleavage, which was partially reduced by caspase-2 specific inhibition while the effect on GO-induced Bax conformational change remained unaltered. In mononuclear cells isolated from AML patients that responded to GO treatment in vitro, processing of caspase-2 was evident, whereas in cells from an AML patient refractory to treatment no such processing was seen. When assessing diagnostic samples from 22 AML patients, who all entered complete remission (CR) following anthracycline-based induction therapy, and comparing patients with long versus those with short CR duration no significant differences in baseline caspase-2 or caspase-3 full-length protein expression levels were found. In summary, we demonstrate that GO triggers caspase-2 cleavage in human AML cells and that the subsequent apoptosis of these cells in part relies on caspase-2. These findings may have future clinical implications.
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| 8. |
- Imreh, Gabriela, et al.
(författare)
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Chromosomal breaks during mitotic catastrophe trigger gamma H2AX-ATM-p53-mediated apoptosis
- 2011
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 124:17, s. 2951-2963
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Tidskriftsartikel (refereegranskat)abstract
- Although the cause and outcome of mitotic catastrophe (MC) has been thoroughly investigated, precisely how the ensuing lethality is regulated during or following this process and what signals are involved remain unknown. Moreover, the mechanism of the decision of cell death modalities following MC is still not well characterised. We demonstrate here a crucial role of the gamma H2AX-ATM-p53 pathway in the regulation of the apoptotic outcome of MC resulting from cells entering mitosis with damaged DNA. In addition to p53 deficiency, the depletion of ATM (ataxia telangiectasia mutated), but not ATR ( ataxia telangiectasia and Rad3-related protein), protected against apoptosis and shifted cell death towards necrosis. Activation of this pathway is triggered by the augmented chromosomal damage acquired during anaphase in doxorubicin-treated cells lacking 4-3-3 sigma (also known as epithelial cell marker protein-1 or stratifin). Moreover, cells that enter mitosis with damaged DNA encounter segregation problems because of their abnormal chromosomes, leading to defects in mitotic exit, and they therefore accumulate in G1 phase. These multi- or micronucleated cells are prevented from cycling again in a p53- and p21-dependent manner, and subsequently die. Because increased chromosomal damage resulting in extensive H2AX phosphorylation appears to be a direct cause of catastrophic mitosis, our results describe a mechanism that involves generation of additional DNA damage during MC to eliminate chromosomally unstable cells.
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| 9. |
- Ivanova, Elena V.
(författare)
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Caspase activation in human neuroblastoma cells: mechanisms and spatiotemporal aspects
- 2015
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis is one of the modes of programmed cell death, in which several members of the caspase family of proteases play the central role. However, activation of apoptotic caspases does not necessarily lead to cell death. Instead, these caspases may mediate, for instance, differentiation or synaptic plasticity, if their activity is restricted in space and time. Such localized caspase activation has been also implicated in the initial stages of neurodegeneration. In order to assess this kind of events at a subcellular level, our research group has previously constructed tau-anchored FRET-based caspase sensors (tAFSs). Here, we demonstrate that localization of tAFSs to the cytoskeleton results in enrichment of the sensors in neuritic processes and enables increased spatiotemporal resolution for live cell imaging of caspase activation, as compared to soluble FRET sensors. This feature is particularly beneficial for investigation of neurodegeneration-related processes.tAFSs were further employed for investigation of caspase activation in neuroblastoma, an extracranial solid pediatric tumor. Tumor necrosis factor-related apoptosis inducing factor (TRAIL) is a promising candidate for cancer treatment due to its ability to selectively trigger apoptosis malignant cells. However, many cancer cells, including neuroblastoma, acquire resistance to TRAIL. Here, we show that in S-type neuroblastoma cell lines, TRAIL resistance is dependent on incomplete activation of apoptotic caspase-3. Sensitization to TRAIL was achieved with protein kinase C (PKC)-inhibiting compounds, suggesting a role for this kinase in blocking the apoptotic response to TRAIL. This effect of PKC could possibly involve stabilization of XIAP, an endogenous caspase inhibitor, as PKC inhibition, in combination with TRAIL treatment, led to downregulation of XIAP.
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| 10. |
- Jangamreddy, Jaganmohan Reddy
(författare)
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Cancer and cancer stem cell targeting agents : A focus on salinomycin and apoptin
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Current cancer treatments involving surgery, radiotherapy, and chemotherapy target the vast majority of cancer cells, but they are only partially effective in eliminating the disease. Failure to eliminate cancer with conventional treatments can lead to recurrence, which usually kills patient. This often occurs when cancer cells develop resistance to cancer drugs or when cancer-initiating cells (cancer stem cells), unaffected by existing treatment procedures, are present. Here, we studied two drugs, salinomycin and apoptin, that exhibit great potential in the future of cancer treatment not only for restricting malignancy, but also in preventing tumor recurrence. Salinomycin is an antibiotic that was used in poultry farming that is now used clinically to target cancer stem cells, and apoptin is a chicken anemia virus-derived protein that is capable of detecting and killing transformed cells. In this study, we delved into the molecular mechanism of salinomycin action leading to cancer cell death. We showed that salinomycin induces autophagy in both cancer and normal primary cells. We further demonstrated that salinomycin promotes mitochondrial fission, thus increasing mitochondrial mass and mitochondria-specific autophagy, mitophagy. Salinomycin-induced cell death was both necrotic and apoptotic as determined by increased release of HMGB1 and caspase-3, -8 and -9 activation. We also found that stress responses of normal and cancer cells to salinomycin differ and this difference is aggravated by starvation conditions. We proposed that a combinational treatment with glucose starvation, or glucose analogues such as 2DG or 2FDG, might enhance the effects of salinomycin on cancer cells while protecting normal cells. We previously reported that apoptin interacts with BCRABL1, a protein that is expressed in patients with chronic myeloid leukemia (CML). We located a minimal region on the apoptin protein that triggers inhibition of downstream BCR-ABL1 signaling effects. This deca-peptide region was tested on patient samples and was shown to effectively kill cancer cells derived from patients, similar to the drug Imatinib. We further show that the apoptin decapeptide is cytotoxic to Imatinib-resistant patient-derived cancer cells. Thus, we identified a novel therapeutic targeting agent that can not only overcome drug resistance, but it can also induce cancer cell death without affecting normal cells.
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