|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
Role of endocytosis and endosomal acidification in TRAIL-induced apoptosis
Hradilová, Naďa ; Anděra, Ladislav (advisor) ; Kovář, Jan (referee)
TRAIL (TNF-related apoptosis inducing ligand) became known for its ability to selectively eliminate cancer cells. This ligand is a member of the TNF (tumor necrosis factor) ligands family and triggers extrinsic apoptotic pathway by binding of its death receptor 4 or 5 (DR4/5), and subsequent formation of death-inducing signalling complex (DISC). This signalling complex is required for successful transmission of apoptotic signal and activation of proximal caspases. However, regulation of the initial steps leading to activation of caspases is still not fully understood. Endocytosis of a TRAIL- DR4/5-DISC complex can be one of modulators of the initiation of extrinsic apoptotic pathway. Recent studies show controversial data documenting that endocytosis of TRAIL receptosomes can in cell type specific manner either positively or negatively influence TRAIL-induced apoptotic signalling. In this study, we focus on the analysis of a role of endocytosis and acidification of endosomal compartments during TRAIL-induced apoptosis in human colorectal cancer cell lines. Our results support the view that both clathrin-dependent endocytosis of TRAIL receptosome and endosomal acidification positively affect activation of caspases during the early stages of TRAIL-induced apoptosis. Inhibition of endocytosis or endosomal...
|
|
|
Role of the mitochondrial pathway in apoptosis induction by taxanes in breast cancer cells
Schmiedlová, Martina ; Kovář, Jan (advisor) ; Anděra, Ladislav (referee)
Apoptosis represents one of the cell death mechanisms which is realized after the application of taxanes in breast cancer cell lines. Apoptosis induction can be principally triggered either by outer or inner pathway. The aim of the diploma thesis is to contribute to the elucidation of role and mechanisms of the inner mitochondrial pathway of apoptosis induction after taxane application (paclitaxel and SB-T-1216) employing a model of breast carcinoma cell lines SK- BR-3 (nonfunctional p53, functional capase-3) and MCF-7 (functional p53, nonfunctional caspase-3). Specifically, we tested the effect of both employed taxanes on mitochondrial membrane potential, ROS level and the expression and localization of proteins regulating inner mitochondrial pathway. Taxane application resulted in mitochondrial membrane dissipation in SK-BR-3 cell line. However, this was not shown in MCF-7 cell line. We found no changes in Bax and Smac/DIABLO expression after taxane application in both tested cell lines. There was a decrease of Bid expression after taxane application in SK-BR-3 line, but not in MCF-7 line. Taxane application did not lead to the translocation of Bax and Bid (tBid) proteins from cytosol to mitochondria in both tested cell lines. Similarly, there was no Smac/DIABLO release from mitochondria to...
|
|
|
Effect of cancer-associated fibroblasts on the survival, proliferation and invasiveness of cancer cells.
Nováková, Gita ; Anděra, Ladislav (advisor) ; Brábek, Jan (referee)
Tumour microenvironment, in addition to cancer cells themselves, represents important structural and functional part of the tumour. Similarly to the normal organs tumour microenvironment comprises several cell types (fibroblasts, immune cells, endothelial cells etc.) and non-cellular components, particularly extracellular matrix. All of them form favourable conditions for the growth, proliferation, protection from the immune system- mediated destruction and nutrition of cancer cells. Cancer associated fibroblasts (CAFs) represent the most abundant cell type of tumour microenvironment. Their origin can be traced to local normal fibroblasts, endothelial cells or epithelial cells and the transition into the CAFs phenotype is influenced with several factors secreted by cancer cells (particularly TGF-β). In contrast to fibroblasts activated during wound healing newly formed cancer associated fibroblasts expressing α-SMA are not subsequently eliminated from the respektive tissue. They persist and produce a number of pro-tumorigenic factors - SDF-1, HGF, IGF-1, IL-6, VEGF, PDGF-C, TGF-β, MMPs etc. CAFs and their secreted factors target several signalling pathways enhancing basic characteristics of the tumour, so called Hallmarks of Cancer. Cancer associated fibroblasts promote proliferation and invasiveness of...
|
|
|
Molecular mechanisms of apoptosis regulation by fatty acids in pancreatic β-cells
Němcová, Vlasta ; Kovář, Jan (advisor) ; Anděra, Ladislav (referee) ; Mělková, Zora (referee)
The incidence of type 2 diabetes is growing rapidly and represents a big threat for the human health care and economy system as well in the 21st century. The association of type 2 diabetes with obesity is apparent and dysfunction and apoptosis of pancreatic β-cells caused by elevated levels of fatty acids in circulation are considered as an important factor contributing to the development of this disease. However, molecular mechanisms that underlie these detrimental effects of fatty acids are only partially understood. The aim of this research project was to contribute to elucidation of mechanisms by which saturated and unsaturated fatty acids regulate viability and apoptosis induction in human pancreatic β-cells in vitro. Employing human pancreatic β-cell line NES2Y, we showed that increased levels of relevant dietary saturated fatty acids (palmitic and stearic acid) induce apoptosis of pancreatic β-cells, in contrast to relevant dietary unsaturated fatty acids (e.g. palmitoleic and oleic acid). We found that stearic acid-induced apoptosis is accompanied by significant activation of caspase-2, -6, -7, -8 and -9, but not by significant activation of caspase-3. Nevertheless, it was not associated with significant cytochrome c release, alteration in PIDD, Fas receptor and Fas ligand expression and...
|
guest ::
