Browsing by Subject "Apoptosis"

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Aktivierung eines neuartigen Apoptose-Signalweges durch den Proteinkinaseinhibitor Staurosporin
(2009) Daubrawa, Merle; Graeve, Lutz
The protein kinase inhibitor staurosporine induces apoptosis via the activation of the intrinsic pathway. First staurosporine was described as a specific PKC inhibitor. Today it is known as a broad range kinase inhibitor and is used as a potent apoptosis inductor. However, the mechanism of the apoptotic effect remains elusive. Furthermore, staurosporine obviously exhibit the potential to eliminate chemotherapy resistant tumors by the induction of a novel intrinsic apoptotic signaling pathway. Different derivatives of staurosporine, e.g. UCN-01, PKC-412 or Enzastaurin are already tested in clinical trials phase I-II for cancer therapy. In the present work it could be shown that overexpression of Bcl-2 does not impede the caspase-dependent induction of apoptosis in J16- and JE6.1-Jurkat T-lymphocytes or in DT40 B-lymphocytes following staurosporine treatment . After generation of apaf-1 -/- DT40 cells it was demonstrated that staurosporine induces apoptosis despite the absence of Apaf-1 and therefore independently of the apoptosome. Together with the generated caspase-9 -/- DT40 cells, caspase-9 was identified as the central effector protein of both staurosporine-induced apoptotic pathways. The involvement of published and putative caspase-9 kinases could not be confirmed by the usage of specific inhibitors. Using phospho-mimicking and phospho-deficient caspase-9 variants, S183 could be identified as an essential phosphorylation site during staurosporine-induced apoptosis. In addition, after treatment with anticancer drugs apoptosome formation was blocked by an N-terminal tag of caspase-9. However, this tag could not prevent staurosporine-induced apoptosis. In further studies the potential role of cathepsines for this novel apoptosis signaling pathway could be analysed by their specific inhibition. In order to investigate the involvement of multiple kinases in this novel apoptotic signaling pathway, combination experiments with specific inhibitors of the respective kinases should be accomplished. Further investigations should clarify whether the influence of S183 on staurosporine-induced apoptosis is based on conformational alteration or on phosphorylation of caspase-9. The generation of additional caspase-9 variants including deltaCARD-caspase-9 or non-cleavable caspase-9 could lead to a deeper understanding of the role of caspase-9 for staurosporine-induced apoptosis. For this purpose caspase-9 -/- DT40 cells and cells reconstituted with different caspase-9 variants could be employed. The phosphorylation pattern of caspase-9 could be determined by mass spectrometric analysis. Xenograft or chorio allantois membrane models were used to investigate if the staurosporine derivative UCN-01 is also able to induce this novel apoptosis signaling pathway in vivo. The identification of both the mechanisms and the effector proteins of this staurosporine-induced apoptotic signaling pathway should provide the opportunity to develop novel agents for the elimination of chemotherapy-resistant tumors.
Eliminierung apoptotischer Zellen durch professionelle Phagozyten: Generierung, Freisetzung und Erkennung des monozytären Attraktionssignals Lysophosphatidylcholin und Bedeutung von Annexin I als Brückenprotein in der phagozytotischen Synapse
(2007) Waibel, Michaela; Graeve, Lutz
The efficient elimination of apoptotic cells by neighbouring cells or professional phagocytes is essential for tissue homeostasis in multicellular organisms. Therefore, the apoptotic cell displays different so-called ?eat-me?-signals on its cell surface that are crucial for its recognition and engulfment. Especially in higher organisms, where the dying cell and the phagocyte are usually not located in immediate proximity, the release of soluble attraction signals is of special importance. Only recently, the phospholipid lysophosphatidylcholine (LPC) could be identified as a central ?find-me?-signal that is generated by the calcium-independent phospholipase A2 (iPLA2)-mediated hydrolysis of phosphatidylcholine. During apoptosis iPLA2 is processed in a caspase-3-dependent fashion. In the present thesis it could be demonstrated that iPLA2 is cleaved directly by caspase-3 and that this processing leads to its activation. The active iPLA2 is essential for the production of the phospholipid-?find-me?-signal LPC in apoptotic cells. However, the observation that overexpression of the active form of iPLA2 alone was not sufficient for the release of the attraction signal from vital cells implied that other apoptotic events might contribute to the generation and export of the ?find-me?-signal LPC. It turned out that the reactive oxygen species-driven oxidation of membrane lipids like phosphatidylcholine is an additional factor that leads to the enhanced production of LPC, probably because oxidized lipids are more susceptible for PLA2-mediated hydrolysis than non-oxidized lipids. Further studies about the detailed export mechanism of LPC revealed that the ATP-binding cassette transporter (ABC)-family member ABCA1 is essential for the release of the attraction signal during apoptosis. Thus, the oxidation of membrane lipids and the ABCA1-driven export of LPC could be identified as important elements of LPC-generation and its subsequent release during apoptosis. After the generation and the release of the attraction signal LPC could be demonstrated in more detail the consequent question arose which receptors might mediate the effects of LPC on the phagocytes. In the present thesis it could be demonstrated that the G-protein-coupled receptor G2A is responsible for the LPC-stimulated migration of monocytic cells. However, the molecular mechanisms that ultimately lead to the LPC-driven, G2A-mediated migration, are not known so far. Accordingly, a participation of other receptors or the existence of further chemotactic signals cannot be ruled out at this point. Moreover, there are some hints for chemotactically active proteins in literature. If these or other factors contribute to the LPC-mediated chemotaxis of monocytic cells is completely unknown and needs to be clarified in future studies. The recognition and internalization of dying cells is mediated by the interaction between different ?eat-me?-signals that are displayed on apoptotic cells, and specific surface receptors on phagocytes. In this scenario, the interaction can be of a direct nature ore rather indirect via bridging molecules. In this context, here it could be demonstrated that the calcium- and phospholipid-binding protein annexin I gets externalized by dying cells independently of the apoptotic stimulus applied, but dependent on the cell type. On the surface of the apoptotic cell, annexin I binds in a calcium-dependent fashion via its annexin-boxes to externalized phosphatidylserine, which represents a central ?eat-me?-signal. Thereby, annexin I is able to stimulate the elimination of these cells by professional phagocytes and thus fulfills the function of a bridging molecule in the phagocytic synapse. In contrast, the receptors that are responsible for the binding of annexin I to phagocytes are not known so far. As a conclusion it can be stated that the phenomena studied in this thesis represent important steps in the process of apoptotic cell elimination. The physiological relevance of apoptotic cell clearance is the fact that apoptosis, in contrast to necrotic cell death, is highly regulated at all stages and usually turns out without any harmful consequences to the organism. If this complex, multistep process is disturbed, non-cleared apoptotic cells can become a source for inflammatory reactions. In different animal models it could be demonstrated that defects in the attraction of phagocytes as well as deficiencies in the recognition and internalization via ?eat-me?-signals and the subsequent degradation of the apoptotic prey can be a reason for the onset of severe autoimmune disorders. In this context, the development of human systemic lupus erythematosus and of chronic arthritis is discussed to be initiated by the inefficient elimination of apoptotic cells.
Role of reactive oxygen species in anti-cancer treatment: Investigations in 2-methoxyestradiol chemotherapy and 5-aminolevulinic acid based photodynamic therapy combined with hyperthermia
(2003) Lambert, Christine; Frank, Jürgen
The thesis deals with two different ROS-generating anti-cancer treatments: chemotherapy with the endogenous estrogen metabolite 2-methoxyestradiol and 5-aminolevulinic acid based photodynamic therapy. Both treatments were investigated with the rat DS-sarcoma model, which can be used in vitro and in vivo. It the first part, it could be shown that 2-methoxyestradiol induces apoptosis in DS-sarcoma cells. Translocation of the pro-apoptotic protein Bax to the mitochondria was identified as initial apoptotic event, followed by a decrease in mitochondrial transmembrane potential and the release of AIF out of the mitochondria. In addition, upregulation of FasL and TNFalpha by 2-ME, two death receptor ligands, was observed. Although, 2-ME administration resulted in activation of caspases, pan caspase inhibitor Z-VAD-FMK could not block 2-ME induced apoptotic cell death pointing to a caspase-independent mechanism. Furthermore, an increase in formation of reactive oxygen species was observed after 2-ME treatment. However, supplementation with different antioxidants could not decrease the toxic effect of 2-ME. This finding may indicate, that reactive oxygen species are not involved in apoptosis induction, rather they are a consequence of mitochondrial damage. In vitro and in vivo combination of 2-ME with another ROS-generating treatment resulted in a synergistic anti-tumour effect. In the second part of the thesis anti-tumour effects of 5-aminolevulinic acid based photodynamic therapy combined with simultaneous hyperthermia was investigated. Analysis of apoptosis associated nuclear changes clearly demonstrated the high efficiency of this treatment regime. Formation of reactive compounds (e.g. ROS, nitrogen monoxide, peroxynitrite) which is mainly responsible for toxicity of PDT, could be assessed in the shape of massive protein nitrosylation in tumours treated with PDT alone or the combined treatment. Detection of decreased amounts of heat shock proteins (HSP70 and HO-1) which protect tumour cells against damaging influences, lowered glutathione levels and reduced MMP-activity indicate an increase in degradation of proteins. This phenomenon may be caused by excessive generation of ROS. Taken together, the presented studies could demonstrate the high benefit of combining 2-ME resp. ALA-PDT with hyperthermia (or other ROS-generating therapies), which make them interesting candidates for future clinical applications.
Steuerungsmechanismen der Spermatogenese beim Eber: Auswirkungen einer GnRH-Immunisierung und anschließender Estradiolinfusion
(2005) Wagner, Anna; Claus, Rolf
Whereas the regulation of spermatogenesis is well known in various species, specific differences, however, exist for the boar, which require species-specific investigation. High concentrations of estrogens are synthesized in the boar testis. It is known, that they play a synergistic role for male behaviour and function of accessory sex glands. Additionally, estrogens in the ejaculate reach the female genital tract and contribute to sperm transport and ovulation in the sow. A possible involvement of estrogens in spermatogenesis, however, was not investigated so far. Similarly, high concentrations of glucocorticoids occur in the tubuli of mature boars. Their regulatory function in spermatogenesis so far is unknown. For clarification, boars were actively immunized against GnRH. GnRH stimulates gonadotropin-release from the pituitary. The evaluation of hormone concentrations in blood plasma after GnRH immunization showed unexpectedly, that the gonadotropin FSH is not influenced, whereas LH- and in consequence steroid- (androgen and estrogen) concentrations were abolished. This offers the possiblity to characterize the selecitve function of estrogens in the presence of FSH. Three groups were evaluated: intact boars (n=5), immunized boars (n=5), 17ß-estradiol infused, immunized boars (n=6). All animals were fitted with indwelling cephalic vein catheters on both sides. One of them was reserved for infusion, the other one for uncontaminated blood sampling. All animals were infused with physiological saline over 7 weeks. For the 17ßEstradiol infused, immunized boars, a defined concentration of estradiol was added to the solution. Blood samples were collected over the whole 7-week period for LH, FSH, testosterone and estradiol. Blood samples were later analysed by established radioimmunological procedures. At the end of the 7-week period, all animals were killed. Tissue samples were fixed for histological examination. Morphological criteria included e.g. tubular diameter, seminiferous epithelial height and number of Leydig cells. Imunocytochemical evaluation was performed for mitosis (Ki-67), apoptosis (TUNEL) and the glucocorticoid receptor. The number of germ cell in the tubuli was reduced by 60% in immunized boars compared to the intact controls. These changes were explained by a more pronounced expression of the glucocorticoid receptor in spermatogonia, followed by the appearance of apoptosis in spermatogonia and primary spermatocytes. In consequence these findings demonstrate for the first time an involvment of glucocorticoids in the quantitative regulation of spermatogenesis. They counteract tubular mitosis by reducing the number of germ cells by apoptosis. In the 17ßestradiol infused group of immunized boars, the infusion led to the restoration of physiological estradiol concentrations (230 pg/ml) in peripheral blood plasma. As expected, androgen concentrations were minimal, so that the specific role of estrogens for spermatogenesis could be clarified. Data from the histological evaluation showed, that the expression of the estrogen receptor alpha occured in spermatogonia, selectively in the mitotic active stage of the seminiferous epithelial cycle leading to a 41-50% rise of germ cell numbers compared to immunized boars. The apoptotic rate in the tubuli of the estradiol infused boars however was not changed compared to immunized boars, so that physiological sperm numbers as they are characteristic for intact boars, were not reached. This study shows for the first time that both glucocorticoids and estrogens play an essential role for spermatogenesis in the boar. They are involved in the regulation of sperm yield by influencing the mitosis-apoptosis equilibrium in the tubuli of boars.
Untersuchungen zur Bedeutung des Sulfonylharnstoffrezeptors 1 für die Modulation von Apoptose durch 17 beta-Estradiol in rekombinanten HEK (Human Embryonic Kidney) 293-Zellen und in pankreatischen beta-Zellen
(2009) Ackermann, Stefanie; Bode, Christiane
The sulfonylurea receptor (SUR) 1 forms the regulatory subunit of pancreatic ATP-sensitive potassium channels (KATP channels) which are essential for triggering insulin secretion in the beta-cell. Insulin secretion is modulated by additional KATP channel-independent pathways and by adaptive variation of beta-cell mass due to apoptosis, proliferation and/or neogenesis of beta-cells. Apoptosis of beta-cells is assumed to be involved in the pathophysiology of diabetes type 1 and 2. Previously it has been shown, that the insulinotropic sulfonylurea glibenclamide and the natural compound resveratrol can induce enhanced apoptosis and that this effect is specifically linked to the expression of SUR1. In the present work, it has been investigated whether there are substances that are more potent in inducing apoptosis than glibenclamide and resveratrol. Thereby the main focus was put on 17beta-estradiol which shows structural and functional analogies to the ?phytoestrogen? resveratrol. Like resveratrol, this naturally occurring estrogen is able to induce apoptosis in different experimental systems. Furthermore, it is known that 17beta-estradiol is able to decrease KATP channel activity in beta-cells acting as a KATP channel blocker. It is still discussed whether 17beta-estradiol directly interacts with KATP channels or whether it binds to so far unidentified ?non-classical? plasmalemmal estrogen receptors which are linked to KATP channels via an intracellular signaling cascade. In heterologous competition experiments, Ackermann et al. (2008) were able to show that 17beta-estradiol is a specific ligand of SUR like glibenclamide and resveratrol. Obviously SUR1 can act as a ?non-classical? estrogen receptor. In the present work it was investigated whether 17beta-estradiol induces apoptosis that is specifically linked to the expression of SUR1. Furthermore, the role of the SUR-isoforms SUR1 and SUR2 and of several SUR-mutants in the induction of apoptosis by 17beta-estradiol was investigated. Therefore, experiments were performed with recombinant HEK293-cells expressing the different isoforms of SUR. Cells that were transfected with empty pcDNA expression vector (pcDNA-cells) were used as control cells. By quantification of different apoptotic parameters such as cell detachment, changes in nuclear morphology as well as increased activity of caspase-3, it was shown that 17beta-estradiol induces specific apoptosis in cells expressing SUR1. In cells expressing the pancreatic isoform SUR1, treatment with 17beta-estradiol resulted in massive apoptosis while cells expressing the cardiac isoform SUR2A or the vascular isoform SUR2B as well as sham-transfected control-cells were less affected. Furthermore, 17beta-estradiol is more potent in inducing apoptosis in cells expressing SUR1 than glibenclamide or resveratrol. The pancreatic KATP channel consists of the regulatory subunit SUR1 and the pore-forming unit Kir6.2. In the present work, it has been proven that this SUR1-dependent effect of 17beta-estradiol was not significantly modified by coexpression with Kir6.2. These data show that apoptosis induced by 17beta-estradiol does not require the existence of functional pancreatic KATP-channels (formed by SUR1 and Kir6.2 subunits). These results provide evidence for an additional function of SUR1 apart from regulating electrical activity of the pancreatic KATP channels. SUR1 might be specifically involved in an adaptive change of the beta-cell mass and could contribute to the regulation of insulin secretion via influencing beta-cell mass. Additional experiments with cells from the clonal beta-cell lines HIT-T15 and RIN-m5F, endogenously expressing SUR1, showed that treatment with 17beta-estradiol can induce apoptosis in these cells. In pancreatic islet cells from mice aged 20-32 weeks, a clear induction of apoptosis after treatment with 17beta-estradiol was observed. Beta-cells of Langerhans also express SUR1 endogenously. Treatment of islet cells from wildtype mice with 17beta-estradiol resulted in intensive changes in nuclear morphology while islet cells from SUR1 knockout (SUR1KO) mice of the same age as well as untreated or solvent-treated islet cells from wildtype and SUR1KO mice did not show any marked signs of apoptosis. In contrast to islet cells from elderly mice aged 20-32 weeks (male or female), clear anti-apoptotic effects were detected in islet cells from young mice aged 5-7 weeks (male or female) after treatment with 17beta-estradiol. In untreated or solvent-treated islet cells from young mice (male or female) apoptosis was measured to a large extent, which was reduced by treatment with 17beta-estradiol. These results provide evidence that age is obviously an important factor which can influence the effect of 17beta-estradiol. The apoptotic effect of 17beta-estradiol in elderly mice as well as the anti-apoptotic effect in younger mice is specific to the expression of SUR1 as it was missing in experiments with islet cells from SUR1KO mice. During pregnancy, plasma concentrations of 17beta-estradiol in humans markedly increase. In the third trimester of pregnancy, 17beta-estradiol concentrations between approximately 50 and 100 nM can be readily achieved. At this point of time, the concentration of maternal serum 17beta-estradiol can be elevated up to more than 100 times compared to serum concentrations during the normal menstrual cycle (follicular phase: approx. 0.1-1.0 nM; luteal phase: approx. 0.5-2.0 nM). Changes in beta-cell mass mediated by 17beta-estradiol might contribute to the etiology of gestational diabetes mellitus (GDM). GDM is defined as glucose intolerance that appears or is first recognized during the last trimester of pregnancy. Estron, another endogenously occurring estrogen, also shows the ability to induce apoptosis in HEK293-cells expressing SUR1 as well as in cells from the SUR1 expressing clonal beta-cell lines HIT-T15 and RIN-m5F. However, the extent of apoptosis after treatment with estron is much lower than after treatment with 17beta-estradiol, although estron differs from 17beta-estradiol only in lacking one hydroxyl group. This hydroxyl group seems to be important for this pronounced SUR1-specific induction of apoptosis by 17beta-estradiol. In the present work, also the role of different SUR-mutants was examined. SUR1(M1289T) is a mutant, in which the amino acid methionine at position 1289 in transmembrane helix 17 (TM17) of SUR1 was exchanged by the corresponding amino acid of SUR2B (threonine). The experiments indicate that the amino acid methionine at position 1289 in TM17 obviously plays an important role in apoptosis which is induced by 17beta-estradiol and is specific for the expression of SUR1. This apoptotic effect after treatment with 17beta-estradiol is abolished by this single mutation. To investigate whether this apoptotic effect after treatment with 17beta-estradiol was linked to a correct function of the nucleotide binding folds, experiments with the mutants SUR1(R1379C) and SUR1(R1379L) were performed. Both mutations are located in the second nucleotide binding fold of SUR1 and result in an enhanced ATP-hydrolysis at the NBFs. These naturally occurring mutations were found in patients with neonatal diabetes with some of them showing a family history in adult-onset type 2 diabetes or GDM. The expression of these mutants in HEK293-cells leads to a much stronger induction of apoptosis than the expression of SUR1. The observation that apoptosis induced by 17beta-estradiol can be influenced by certain mutations of SUR could be of particular importance for the pathophysiology of diseases like cancer or diabetes. The enhanced activity of caspase-9 in cells expressing SUR1 after treatment with 17beta-estradiol suggests that the mitochondrial pathway might play a major role in this apoptotic process.
Up-regulation of fibroblast growth factor 23 gene expression in UMR106 osteoblast-like cells with reduced viability
(2021) Münz, Sina; Feger, Martina; Edemir, Bayram; Föller, Michael
Fibroblast growth factor 23 (FGF23) controls vitamin D and phosphate homeostasis in the kidney and has additional paracrine effects elsewhere. As a biomarker, its plasma concentration is associated with progression of inflammatory, renal, and cardiovascular diseases. Major stimuli of FGF23 synthesis include active vitamin D and inflammation. Antineoplastic chemotherapy treats cancer by inducing cellular damage ultimately favoring cell death (apoptosis and necrosis) and causing inflammation. Our study explored whether chemotherapeutics and other apoptosis inducers impact on Fgf23 expression. Experiments were performed in osteoblast-like UMR106 cells, Fgf23 gene expression and protein synthesis were determined by qRT-PCR and ELISA, respectively. Viability was assessed by MTT assay and NFκB activity by Western Blotting. Antineoplastic drugs cisplatin and doxorubicin as well as apoptosis inducers procaspase-activating compound 1 (PAC-1), a caspase 3 activator, and serum depletion up-regulated Fgf23 transcripts while reducing cell proliferation and viability. The effect of cisplatin on Fgf23 transcription was paralleled by Il-6 up-regulation and NFκB activation and attenuated by Il-6 and NFκB signaling inhibitors. To conclude, cell viability-decreasing chemotherapeutics as well as apoptosis stimulants PAC-1 and serum depletion up-regulate Fgf23 gene expression. At least in part, Il-6 and NFκB may contribute to this effect.