Browsing by Subject "DNA repair"

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Bedeutung der c-Abl-Aktivität für die Reaktion auf DNA-Schädigung und für die genetische Stabilität Bcr-Abl-negativer Zellen
(2011) Fanta, Silke; Aulitzky, Walter E.
The launch of Imatinib (Glivec®, Gleevec®, STI571) in August 2001 was an important advancement in the therapy of chronic myeloid leukemia (CML). The small-molecule inhibitor directly targets the oncogenic tyrosine kinase Bcr-Abl, which has been identified as the central cause for the development of CML. Treatment with Imatinib is the gold standard in the therapy of CML. However, taking the current state of research, an elimination of the malignant Bcr Abl-positive clone cannot be achieved by treatment with Imatinib. Thus, long-term or even lifelong treatment of patients is necessary. As a consequence, it is of great interest to clarify the biological effects of Imatinib on physiologically normal cells. Previous studies of the group showed that Imatinib treatment of Bcr Abl-positive cells leads to a decreased mutation frequency following DNA damage. Within the scope of the present work, evidence for significantly enhanced mutation rates after DNA damage in non-cancerogenic primary human lymphocytes (PBMC) and murine hematopoietic cell lines (32D and BaF3) after Imatinib treatment was obtained for the first time. Thus, Imatinib treatment of Bcr Abl-negative cells shows opposite effect compared to Bcr Abl-positive cells. It was therefore proven that the Imatinib-related inhibition of Bcr Abl as well as the off-target effects in Bcr Abl-negative cells play an important role in the genetic stability. To determine whether an Imatinib-mediated inhibition of c Abl activity is responsible for effects independent of Bcr Abl, genetic c Abl models were used to assess stress-induced mutation frequency. To this, we employed c Abl-knockout-MEFs (embryonic mouse fibroblasts), which were retransfected with wild type c Abl and a kinase-deficient form, respectively. After DNA damage, there was a significant increase in mutation frequency in the kinase-deficient cells (MEF Abl-KD) when compared to the c-Abl wild type (MEF Abl-wt) cells. Consequently, c-Abl activity is of great importance for the maintenance of genetic stability. Several factors can result in an increased mutation frequency in cells. Examples include altered cell proliferation, impaired DNA repair mechanisms or a delayed induction of cell death. In the latter case, DNA damage is not adequately repaired and passed to the daughter cells. In this study, different hematopoietic cell lines were used to show that neither the pharmacological nor the genetic inhibition of c-Abl activity has an influence on induction of cell death, division rate, cloning efficiency and cell cycle distribution. To investigate how far Imatinib influences the kinetics of DNA strand break repair after irradiation, alkaline comet assays were performed. Imatinib treatment of cells had no influence on induction of strand breaks or constitutive strand breaks prior to irradiation. However, cells treated with Imatinib exhibited a significantly delayed repair of DNA strand breaks. This delay was shown in the same manner in hematopoietic cell line models and in primary human lymphocytes, which were treated with Imatinib as well as with Dasatinib, a second generation Abl-inhibitor. Cell line models with different forms of c-Abl were used to provide evidence that this effect is caused by inhibition of the c-Abl kinase activity. The delayed repair of DNA strand breaks was also seen in cells with a kinase-deficient form of c-Abl (MEF Abl KD). But treatment with Imatinib had no effect on the kinetics of DNA repair in cells that expressed an Imatinib-resistant form of c Abl (c Abl T315I). Double- (DSB) as well as single-strand breaks (SSB) are determined in an alkaline comet assay. By applying neutral conditions, this assay can be modified to exclusively analyze DSB repair. As expected, there was a significantly lower induction of DSB after irradiation when compared to the occurrence of SSB. However, Imatinib did neither influence the induction nor the kinetics of DSB repair. Both pulsed-field gel electrophoresis and the quantification of gamma-H2AX were used to confirm that Imatinib does not affect DSB repair. Rather, the delayed repair kinetics are exclusively caused by an Imatinib-dependent interference with SSB repair. Extensive investigations of the molecular signaling pathways of DNA damage repair show that inhibition of c Abl activity does not affect ATM-Chk2-p53 or ATR-Chk1 signaling. Poly(ADP-ribosyl)ation of proteins is an early event in the processing of the SSB repair. This modification of proteins by addition of long and branched poly(ADP-ribose) chains (PAR) is an essential part of the SSB repair and base excition repair (BER). Both the synthesis and the cleavage of PAR is mediated by the kinases PARP-1 (poly(ADP-ribose) polymerase-1) and PARG (poly(ADP-ribose) glycohydrolase). This activity was determined by quantification of PAR and the percentage of cells, which were PAR-positive at a certain time. Possible effects of an Imatinib-induced inhibtion of c-Abl on poly(ADP-ribosyl)ation were investigated. To this, a method for the measurement of PAR events on a single-cell level was established. Poly(ADP-ribose) residues were marked with a PAR-specific antibody and detection followed by means of a fluorochrome-conjugated secondary antibody. The specificity of the method was proven unequivocally by a complete loss of signal when a specific PARP inhibitor (PJ34) was applied prior to irradiation-induced ribosylation. The advantage of this method is that the simultaneous determination of the DNA content in every cell allows the analysis of ribosylation events in correlation with cell cycle distribution. Based on these experiments it was found that in Imatinib-treated cells both the constitutive and the irradiation-induced poly-ribosylation are significantly enhanced. Furthermore, irradiation does not result in poly-ribosylation of all cells at a certain time: A subpopulation of cells, presumably those in the G0 resting phase, remain PAR-negative before and after irradiation. Thus, a novelty of the work at hand lies in the correlation of ribosylation events and cell cycle distribution before and after DNA damage. In this context, the central role of the Imatinib-mediated inhibition of c-Abl could also be established. The inhibited kinase activity of c-Abl seems to cause a delayed degradation of PAR. This is either caused by decreased activity of the PARP-1 antagonist PARG or by increased activity of PARP-1 itself. A disturbance of the spatially and temporally tightly modulated synthesis and degradation of PAR may lead to a prolonged interaction of PARP-1 with proteins related to SSB repair or BER, e.g. XRCC1 and DNA polymerase beta, thus resulting in the observed delay in DNA damage repair. The present study provides new insights into the impact of Imatinib on Bcr Abl-negative cells. The obtained in vitro data suggest that long-term treatment with c-Abl inhibitors may be associated with an increased likelihood of secondary neoplasias. Despite the outstanding success in Imatinib treatment of CML patients in the chronic phase, the complete elimination of the malignant clone should be the primary goal of the treatment of Bcr-Abl-positive leukemias.
Einfluss eines Glukoseentzugs auf die Strahlenempfindlichkeit von Tumorzellen und Normalzellen
(2018) Ampferl, Rena; Dittmann, Klaus
Radiotherapy is a major pillar of cancer treatment. However, the maximal dose that can be applied to a tumor is limited by side-effects of the irradiated normal tissue. Therefore, to improve treatment success, it is of significant interest to develop new treatment strategies that selectively enhance the cytotoxic effect of radiation in tumor cells while sparing healthy tissue. For this purpose, it is necessary to exploit differences between tumor cells and normal cells. Thus, tumor cells are characterized by metabolizing glucose preferentially to lactate regardless of the availability of oxygen (Warburg effect, aerobic glycolysis), while normal cells oxidize most of the glucose in the mitochondria if oxygen is present. Because the Warburg effect only produces low amounts of ATP per molecule of glucose when compared to mitochondrial glucose oxidation, tumor cells rely on high glucose supply. Hence, it was the aim of this study to investigate whether a glucose starvation during radiotherapy, which requires energy-dependent repair of DNA damage, is an appropriate strategy to selectively enhance radiosensitivity of tumor cells, but not of normal cells. It was shown that glucose starvation inhibited proliferation of the tumor cell lines A549 and FaDu, but not that of the normal fibroblasts HSF7. Moreover, deprivation of glucose induced cell death selectively in tumor cells, which occurred mainly via necrosis. Combining glucose starvation with radiotherapy led to selective radiosensitization of both tumor cell lines, which was accompanied by impaired repair of radiation-induced DNA double-strand breaks (DNA DSBs). In this context, it turned out that in tumor cells glucose is essential for the late stage of DNA DSB repair starting from 13 h after irradiation. Furthermore, an inhibition of radiation-induced histone acetylation as well as KAP1 phosphorylation could be observed in tumor cells following glucose starvation, indicating an impairment of radiation-induced chromatin relaxation. Because opening of the chromatin structure is particularly important for the repair of DNA DSBs within heterochromatin and because these DSBs are the ones that are repaired at late time points after irradiation, it can be assumed that in tumor cells glucose starvation mainly impairs the repair of heterochromatic DNA DSBs. Further investigations revealed that in tumor cells glucose starvation does not cause lack of nuclear acetyl-CoA, which is the substrate for the acetylation of histones, and therefore this could be excluded as cause of the observed inhibition of histone acetylation. However, it is known that the histone deacetylase Sirt1 is activated in response to glucose starvation. Histone deacetylation by Sirt1 could counteract radiation-induced histone acetylation, thus impairing chromatin relaxation as well as repair of DNA DSBs after irradiation. In fact, it was shown that inhibition of Sirt1 by sirtinol can partly abrogate the impaired repair of radiation-induced DNA DSBs that was observed in tumor cells under glucose-free conditions. However, the inhibitory effect of glucose starvation on DNA DSB repair in tumor cells could not only be observed under glucose-free conditions. Thus, reducing the glucose concentration to 0.5 g/l was enough to impair DSB repair following irradiation to the same degree as after total deprivation of glucose. Furthermore, it turned out that under glucose-free conditions DNA DSB repair in tumor cells was promoted by autophagy already after irradiation with 2 Gy. Finally, it was shown that, in addition to DNA DSB repair, also tumor metabolism is influenced by glucose starvation. Thus, deprivation of glucose impaired the radiation-induced switch of glucose metabolism that was characterized by increased aerobic glycolysis and decreased mitochondrial glucose oxidation, and this can also contribute to radiosensitization of the cells. In contrast to tumor cells, glucose starvation neither caused radiosensitization nor impaired the repair of radiation-induced DNA DSBs in normal fibroblasts. Moreover, in these cells, glucose starvation had no influence on histone acetylation and KAP1 phosphorylation after irradiation. These results demonstrate that glucose starvation is an appropriate in vitro strategy to selectively sensitize tumor cells to radiotherapy without influencing the radiosensitivity of normal cells.
Investigations on the mechanisms of sterilization by non-thermal low-pressure nitrogen-oxygen plasmas
(2011) Roth, Stefan; Hertel, Christian
Plastic based materials are increasingly used for packaging of pharmaceuticals (especially biologicals), food or beverages and production of medical devices. Their heat sensitivity requires safe and efficient non-thermal methods for decontamination. Plasma technology has the potential to provide a suitable means since it works at low temperatures and ? in contrast to conventional methods like application of ionizing radiation or ethylene oxide exposure ? is safe to operate, is free of residues and does not alter the bulk properties of the materials. Plasmas can generate various agents potentially active in decontamination like ultra-violet (UV) radiation, radicals and other reactive particles. To acquire an approval for plasma technology as a novel sterilization method, its process safety has to be proven. The research community has proposed hypotheses and models on its mechanisms of action, which are at least partially speculative. Still little is known about the details of the biologic effects of the combination of the various plasma agents on the components of microbial cells or spores. Especially, the question remains open which components of a cell or spore are the primary targets, and which of the agents are most effective in the inactivation process. The acquisition of such knowledge is necessary to identify parameters suitable to control, monitor, and assess the safety of plasma sterilization processes. The aims of the presented work are to elucidate which components of a cell or spore are the primary targets in low-pressure plasma sterilization, and which of the putative agents contained in the plasma are most effective in the inactivation process. To accomplish this, in the presented work suitable microbiological methods were established and the inactivation of bacterial spores and cells and fungal conidia by microwave induced low-pressure low-temperature nitrogen-oxygen plasmas was investigated. Moreover, two strategies were pursued that have hitherto not been applied in published plasma sterilization studies: (i) Using spores of Bacillus subtilis mutants to identify structural components serving as targets for sterilization with plasma and (ii) characterizing the response of Deinococcus radiodurans R1 cells to plasma treatment and identify repair processes during recovery from plasma induced damages in viable cells. Plasmas producing a maximum of UV emission were most effective in inactivating bacterial cells and spores. The inactivation followed a biphasic kinetics consisting of a log-linear phase with rapid inactivation followed by a slow inactivation phase. A continuous model fit was applied to the experimental data allowing reliable calculation of decimal reduction values for both phases. Cells of D. radiodurans were found to be more resistant than spores of B. subtilis. For B. subtilis spores, in the course of plasma treatment damage to DNA, proteins and spore membranes were observed by monitoring the occurrence of auxotrophic mutants, inactivation of catalase (KatX) activity and the leakage of dipicolinic acid, respectively. Spores of the wild-type strain showed highest resistance to plasma treatment. Spores of mutants defective in nucleotide excision repair (uvrA) and small acid-soluble proteins (ΔsspA ΔsspB) were more sensitive than those defective in the coat protein CotE or spore photoproduct repair (splB). Exclusion of reactive particles and spectral fractions of UV radiation from access to the spores revealed that UV-C radiation is the most effective inactivation agent in the plasma, whereby the splB and ΔcotE mutant spores were equally and slightly less sensitive, respectively, than the wild-type spores. The extent of damages in the spore DNA as determined by quantitative PCR correlated with the spore inactivation. Spore inactivation was effectively mediated by a combination of DNA damage and protein inactivation. DNA was identified to be the primary target for spore inactivation by UV radiation emitted by the plasma. Coat proteins were found to constitute a protective layer against the action of the plasma. For the investigation of the recovery from plasma-induced damages, cells of D. radiodurans R1 were subjected to short plasma treatments with various plasmas. A part of the survivors was sublethally injured as determined by their ability to form colonies on standard medium but not on stress medium and by the observation of a prolonged lag phase. Incubation of the cells in a recovery medium after plasma treatment allowed a part of the survivors to recover their ability to grow on stress medium. This recovery strongly depended on transcriptional and translational processes and cell wall synthesis, as revealed by addition of specific inhibitors to the recovery medium. Genes involved in DNA repair, oxidative stress response and cell wall synthesis were induced during recovery, as determined by quantitative RT-PCR. Damage to chromosomal DNA caused by plasma agents and in-vivo repair during recovery was directly shown by quantitative PCR. Plasmas with less UV radiation emission were also effective in killing D. radiodurans cells but resulted in less DNA damage and lower induction of the investigated genes. The response of D. radiodurans to plasma indicated that DNA, proteins and cell wall are primary targets of plasma, whose damage initially leads to the cells' death. Protein oxidation was more important for the killing of D. radiodurans cells than of B. subtilis spores. Thus, the plasma process parameters must regard the expected contaminating biological material in order to obtain a high-level sterilization. The results provide new insight into the interaction of non-thermal low-pressure plasmas with microorganisms. This knowledge supports the definition of suitable parameters for novel plasma sterilization equipment to control process safety. For example, monitoring the UV intensity below 280 nm and spectrometric online measurement of bands related to excited reactive gas particle species during the process is recommended.
Nuclear activation of proteasome in oxidative stress and aging
(2009) Catalgol, Betul; Grune, Tilman
Poly(ADP-ribosyl)ation reactions are of interest in recent years and they take place in DNA repair in different processes especially following oxidative nuclear damage. Proteasomal reactions also take place in repair following oxidative nuclear damage with the degradation of oxidized histones. Antitumor chemotherapy is generally believed to act via the oxidation of nuclear material in the tumor cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs. The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the nucleus. Tumor cells are supposed to have a higher nuclear proteasome activity than do nonmalignant cells. Poly(ADP-ribosyl)ation reactions take place in the tumor cells as a consequence of chemotherapy. Such a reaction might occur with the 20S proteasome ?which is known to increase the activity- and also with histones ?which is firstly shown to decrease the degradation in this study. After hydrogen peroxide treatment of HT22 cells, degradation of the model peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increased accompanied by an increase in PARP-1 mRNA expression. In the recovery of the level of protein carbonyls, single strand breaks and 8-OHdG, proteasome and PARP-1 were shown to play a role together. This was tested with inhibitor treatments. The proteasomal activation following poly(ADP-ribosyl)ation of proteasome and the decrease in poly(ADP-ribosyl)ation of histones and increase in the proteasomal degradation of histones following H2O2 treatment confirmed our hypothesis. The second part of the thesis shows the changes in PARP-1 and proteasome in different aged fibroblasts with population doublings 19, 36, and 56. The nuclear protective mechanisms were shown to be effected during the senescence process. PARP-1 protein amount decreased whereas there was no change in proteasome amount. PARP activation following H2O2 treatment increased only in young and middle aged cells. In the nuclear extracts of young and old cells, poly(ADP-ribosyl)ation potentials were tested with NAD+ addition into the reaction. In addition to that active proteasome and PARP enzymes were added into the reaction and proteasome activity was measured. With active PARP, proteasome activity was increased both in young and old cells whereas there was no increase in old cells without PARP addition. These results show that proteasome activation is mainly limited by PARP activity. Taken together all results demonstrate the importance of PARP mediated proteasome activation in the repair of oxidatively damaged chromatin.