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Programmed necrosis: its activation, regulation and role in cellular physiology.
Rytířová, Markéta ; Anděra, Ladislav (advisor) ; Holzerová, Kristýna (referee)
Cell death as the final stage of cell existence can be either triggered accidently or it can result from the activation of specific controllable signalling pathways. Regulated or programmed cell death can be induced by number of extrinsic or intrinsic stimuli under both physiological and pathological conditions. For a long time, caspase-dependent apoptosis has been considered as the only form of programmed cell death. However, in the last 10 years it has been shown and proofed that also necrotic cell death, formally considered as random and uncontrollable cell death, may also proceed in controllable manner with specific signalling pathways and features. Among the signalling pathways associated with the programmed necrosis belong activation of RIP1/RIP3 kinase-containing necrosome, then cyclophilin D assisted mitochondrial permeabilization or response to oxidative stress. To the most studied signal transduction pathways associated with the iniciation of programmed necrosis belongs TNF-mediated activation of RIP1/RIP3-dependent necroptosis. Programmed necrosis plays an important role in various physiological and pathological processes, for example in ischemic-reperfusion disorders, diseases of the eye, skin, gastrointestinal system, and also can be triggered in cancer therapy. Key words Cell death,...
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Srovnání indukce a regulace autofagocytózy v proliferujících a senescentních nádorových buňkách
Pešina, František ; Anděra, Ladislav (advisor) ; Rudolf, Emil (referee)
Autophagy, senescence and apoptosis are tightly linked processes which together determine the fate of cells in response to various stresses. There is ample evidence supporting the notion that senescent cells are highly dependent on autophagy and this process is here much more intensive than in nonsenescent cells. Autophagy may to some extent compensate increased energetic and metabolic demands of senescent cells and also helps with removal of toxic products such as oxidized proteins, protein aggregates and damaged organelles resulting from an overloaded metabolism of some senescent cells. In addition, some studies reported the need of autophagy for the adoption of senescent phenotype. However, there are also studies with seemingly contradictory results claiming that increased autophagy prevents or delays cellular senescence. Relationship of autophagy to apoptosis is similarly ambivalent. Whereas intact autophagy is necessary for the cell, while slightly increased autophagy still has a rather positive impact, excessive autophagy may lead to degradation of critical components necessary for cell function and survival and can trigger one of the modes of programmed cell death. In the first part of this work, we focused on the analysis of autophagic response in senescent and proliferating pancreatic...
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Charakterizace vlivu senescence na indukci a regulaci smrti nádorových buněk
Nováková, Gita ; Anděra, Ladislav (advisor) ; Truksa, Jaroslav (referee)
4 Abstract Senescence is a specific cell state distinquished by cessation of cell division and proliferation and changes in gene expression. Normal cells enter senescence after distinct number of cell divisions or in case of an unrepairable damage. Senescence in cancer cells can be induced by subliminal stress as sublethal treatment with certain drugs. Senescent cancer cells persist in the tissue and may secrete a number of factors and nutrients affecting surrounding cells. Senescence can thus change the response of cancer cells to various apoptogens during cancer therapy. In this study, we focused on the elucidation of presumed differences between normal proliferating and senescent cancer cells in their response to selected apoptogens. Implementing bromodeoxyuridine (BrdU)-mediated replication stress in cancer cells derived from pancreatic (PANC-1) or mesothelioma (H28) tumors, we efficiently forced these cells to acquire senescent phenotype. We document that these senescent cells gain higher resistance to combined TRAIL and homoharringtonine (HHT) treatment and enhance sensitivity to other apoptogens such as FasL, camptothecin and mVES. These cells also showed increased expression of anti-apoptotic protein c-FLIP in senescent cells and changes in the expression of some Bcl-2 family proteins....
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Cell death-regulating micro RNAs and their role in the development and pathological processes.
Běhounek, Matěj ; Anděra, Ladislav (advisor) ; Seifertová, Eva (referee)
MicroRNAs are small protein non-coding, ~ 22 nucleotides long dsRNAs. Their main task is suppression of gene expression via removal/destabilization of mRNA or its targeting to degradation. These small molecules play an important role in the regulation of many cellular processes and have been found to affect expression of more than 30% of human genes. Among the processes affected or regulated by miRNAa belongs also programmed cell death. Although this work is mainly focused on the analysis and characterization a role of distinct miRNAs in the regulation of apoptotic cell death, miRNAs can also participate in the regulation of autophagic cell death or programmed necrosis. MiRNA can enhance cellular sensitivity to apoptosis by suppressing the expression of death receptor genes, but can also drive cells to apoptosis by regulating expression of anti-apoptotic protein Bcl-2. In many different organisms were already discovered and described thausends of micro RNAs anddozens of them participate in the regulation of cell death. Poor or impaired function of miRNAs and related disturbance in apoptotic signaling could lead to a number of pathological processes as tumorigenesis or disturbances in tissue development and homeostasis. . Understanding how miRNA functions in cell death and possible practical...
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The significance of autophagy and its communication with the apoptotic machinery for cellular survival or cell death
Pazour, Vítězslav ; Anděra, Ladislav (advisor) ; Černý, Jan (referee)
Autophagy is a cellular proces, taht allows degradation of a portion of cytoplasm, protein aggregates or entire organelles. Major function of autophagy is the maintainance of cellular homeostasis, the protection against stress and mobilization of internal resources. However, autophagy also has a role in imunity, development and differentiation. Autophagic signaling can interact with apoptotic machinery at several levels via regulatory proteins of both pathways or via mutual degradation or cleavage of the components of both pathways. Autophagy can communicate with both extrinsic and intrinsic pathways of apoptosis. Under certain circumstances can autophagy by itself also induce cell death. Autophagic cell death called also programmed cell death of type II is accompanied by massive vacuolization and lysosomal autodestruction of the affected cell. Autophagic cell death was documented during Drosophila development but also in mammalian cells. Autophagy also play importamt role in tumorogenesis, where it can either protect tumor cells against various stresses or it can contribute to their death. Further research of autophagic signaling and mechanisms of communication between autophagy and apoptosis may ont only extend our knowledge on these essential processes but can also contribute to cancer therapy. Powered...
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Charakterizace TRAILem indukované, receptor-specifické signalizace v nádorových buňkách.
Peterka, Martin ; Anděra, Ladislav (advisor) ; Rohlena, Jakub (referee)
TNF-related apoptosis-inducing ligand (TRAIL) is a member of TNF family expressed mainly by hematopoietic cells. TRAIL brought significant attention mainly for its ability to trigger apoptosis in a number of cancer cells. In addition to apoptosis, TRAIL can induce several other signaling pathways such as activation of MAP kinases or canonical NF-B signaling. Human TRAIL can bind to five receptors but only two of them (death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5) can trigger TRAIL-mediated apoptotic and non-apoptotic signaling in target cells. Both receptors are ubiquitously expressed on normal and cancer cells, but the relative contribution of DR4 and DR5 to TRAIL-induced signaling is not well known. Using DR4/DR5-specific variants of TRAIL, we examined how individual receptor contributes to the induction of apoptosis and NF-B, JNK, p38, ERK1/2 and TAK1 signaling pathways in selected colorectal cells. We found that in DLD-1 cells, apoptosis and activation of JNKs are mainly mediated by DR4-selective ligand. In TRAIL-resistant HT-29 cells, we show that though DISC formation and activation of caspase-8 proceeds mainly via DR4-specific signaling, activation of NF-B pathway is mainly triggered by DR5 selective ligand. In other cells and analyzed signaling pathways both receptor-specific ligands triggered very...
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Mechanism, regulation and use of TRAIL-induced apoptosis in cancer cells
Horová, Vladimíra ; Anděra, Ladislav (advisor) ; Truksa, Jaroslav (referee) ; Živný, Jan (referee)
Tumour necrosis factor-Related Apoptosis Inducing Ligand (TRAIL), a membrane- bound ligand from the TNF family, has attracted significant attention due to its rather specific and effective ability to induce apoptotic death in various types of cancer cells via binding to and activating its pro-apoptotic death receptors (DRs). However, a significant number of primary cancer cells often develop resistance to TRAIL treatment, and the signalling platform behind this phenomenon is not fully understood. In the first paper we focused on the influence of endosomal acidification. Upon blocking endosomal acidification by the vacuolar ATPase (V-ATPase) inhibitors bafilomycin A1 (BafA1) or concanamycin A (CCA) we observed a significantly reduced initial sensitivity of several, mainly colorectal, tumour cell lines to TRAIL-induced apoptosis. In cells pre- treated with these inhibitors, the TRAIL-induced processing of caspase-8 and the aggregation and trafficking of the TRAIL-receptor complexes were temporary attenuated. The cell surface expression of TRAIL receptors and their TRAIL-induced internalization were not affected by V-ATPase inhibitors. NF-κB or MAP kinase signalling from the activated TRAIL receptors remained unchanged, and neither possible lysosomal permeabilization, mitochondrial amplification loop...
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Apoptosis of tumor cells : role of TRAIL and caspase 10
Truxová, Iva ; Živný, Jan (advisor) ; Anděra, Ladislav (referee)
One of the key features of cancer cells is the ability to escape programmed cell death (apoptosis). As a mechanism of apoptosis inactivation in cancer cells, somatic mutations of pro-apoptotic genes have been reported in many cancers. Caspase 10 is an initiator caspase whose physiological function remains poorly understood. Also the ability of caspase 10 to substitute for caspase 8 in the death receptors apoptotic pathway is still controversial. However, the fact that some of the mutations found in CASP10 gene was associated with apoptosis defects (79, 81) suggest that caspase 10 could be also important in apoptosis initiation. In our lab, there was found a heterozygous mutation in CASP10 gene of Jurkat (human T-acute lymphoblastic leukemia) clone resistant to TRAIL (J-TR1). This mutation influence the amino acid composition close to the active site of the enzyme. The aim of this thesis was to confirm the mutation by ARMS-PCR and to determine if an overexpression of normal (unmutated) or mutated caspase 10 D in TRAIL sensitive and/or TRAIL resistant Jurkat cells (J-WT and/or J-TR1) will influence TRAIL induced apoptosis. Mutation was confirmed. We created J-WT and J-TR1 stable clones transfected by vector with unmutated or mutated CASP10 D (CASP10 D WT or CASP10 D MUT). CASP10 D MUT overexpression in J-WT...
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Type of cell death and superinfection exclusion phenomenon in vaccinia virus-infected cells
Lišková, Jana ; Mělková, Zora (advisor) ; Anděra, Ladislav (referee) ; Němečková, Šárka (referee)
Vaccinia virus (VACV) was formerly used in the eradication campaign against smallpox. VACV infection causes lysis of most cell types, including epithelial ones, which is equivalent to necrosis. However in our laboratory, we have previously detected activation of caspases during infection of HeLa G and BSC-40 epithelial cell lines with VACV, a typical sign of apoptosis. In this thesis, the type of cell death in HeLa G and BSC-40 cell lines infected with VACV strain WR was further studied and the activation and activity of caspases in the infected cells was characterized. In HeLa G cells infected with VACV strain WR, apoptosis was identified, as demonstrated by condensed nuclei, activity of caspase-3 and cleavage of death substrates. Additionally, activation and activity of caspase-2 and -4 was detected in infected HeLa G cells. In infected BSC-40 cells, neither apoptosis nor caspase activity were observed, but activation of caspase-2 and 4 was detected in these cells also. Finally, cleavage of procaspase-3 and -12 was detected in infected cells of both cell lines. Vaccination strains Praha and Dryvax induced apoptosis in both HeLa G and BSC-40 cells, as was demonstrated by the apoptotic morphology of nuclei and by the cleavage of PARP, substrate of the executioner caspases. Our results suggest that...
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Molecular and functional characterization of the death receptor 6
Klíma, Martin ; Anděra, Ladislav (advisor) ; Živný, Jan (referee) ; Kovář, Marek (referee)
Death receptor-6 (DR6/TNFRsf21/CD358) is a receptor from the TNFR superfamily that likely participates in the regulation of proliferation and differentiation of T- and B-lymphocytes and neural cells. The 655-amino acid human DR6 is a type I transmembrane protein containing four cysteine-rich domains in its extracelular part and a death domain followed by the CARD-like region in its cytoplasmic part. Overexpression of DR6 in some cell lines leads to apoptosis, and/or to activation of nuclear factor NF-κB and stress kinases of the JNK family. In the first part of our work we focused on molecular characterization of DR6, including the analysis of its posttranslational modifications. We found that DR6 is an extensively posttranslationally modified protein including S-palmitoylation and both N- and O-glycosylation. Six N-glycosylation and one S-palmitoylation sites were precisely mapped to appropriate asparagines and cysteine respectively. The juxtaposed linker region (between cystein-rich domains and the transmembrane part), which also contains Ser/Thr/Pro-rich region with clustered putative O-glycosylation sites, is required for the plasma membrane localization of DR6. N-glycosylation, but interestingly not S-palmitoylation, may play a role in targeting of DR6 into detergent-resistant...
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