Browsing by Subject "Inhibitor"

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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.
Grundlagenuntersuchungen zur Wirkung neuartiger Ureaseinhibitoren in der Nutztierhaltung
(2008) Reinhardt-Hanisch, Annett; Hartung, Eberhard
Due to the agreements held by Germany in international and national programmes to reduce climate and environmental deterioration particularly in relation to ammonia emissions from livestock housing, additional measures have to be taken in order to ensure that international and national objectives will be achieved. Within the framework of a BMBF-funded interdisciplinary research project, new types of urease inhibitors had been tested for application in animal housing. The present work shows the necessary basic studies, which formed the basis for the investigations under conditions similar stall conditions and in practice stall in the second part of the project (LEINKER, 2007). The main objective of the present work was to investigate the effect of different urease inhibitors on urea hydrolysis, and thus to quantify the ammonia release of urea applied on cattle slurry under standardized, reproducible and controlled laboratory conditions and to investigate possible influencing factors (Pre-screening). Subsequent objectives result in the following: development of an appropriate measuring system and implementation of a standardized test, study of various urease inhibitors at different substrate temperatures and inhibitor concentrations, selection of the urease inhibitor which has the best reduction effect, and quantification of other factors influencing the effect of the selected urease inhibitor. In order to assess the inhibitor effect, the protonated ammonium and the available ammonia in slurry should be detected by means of conducting slurry analysis. In addition, the first indications of a long-term effect of the selected urease inhibitor, its impact on biogas accumulation, and its effect on liquid swine manure should be derived. In order to carry out the investigations, a water-tank measuring system had been configured and validated. The 28 measuring tanks of the measurement system, where each tank was filled by 2 l of slurry, worked according to the principle of dynamic chamber and were kept at constant temperature using a water bath. A gas analysis based on NDIR spectroscopy had been used in order to sustain continuous measurement of ammonia concentrations in the measuring device. The standardized test, applied in the main experiments, had been developed in the preliminary experiments. Among the five pre screened urease inhibitors (four new active agents + reference NBTPT active ingredient), the urease inhibitor D is selected for further investigations in the project, as this is the best inhibitor having significant reduction effect, which was in general dependent on the substrate temperature and the inhibitor concentration in terms of ammonia release. For an effective reduction of ammonia release, it seems useful to deploy more often smaller quantities of the inhibitor. The urease inhibitor D showed a significant reduction effect regardless of the origin and the layer thickness of the respective liquid manure. In comparison with the urease inhibitor D treated variants, more urea is significantly converted to ammonia and ammonium-nitrogen at a layer thickness of 4 mm than at a layer thickness of 90 mm. Apart from the ammonia release, the resulting nitrogen balance of all the tests showed no indication of further significant gaseous nitrogen losses, e.g. as nitrous oxide. In the experiments, no significant influence of urease inhibitor D had been detected on the (urea independent) basic release of ammonia from the respective liquid manure resulting from the degradation of organic substances. In practice, this means that the use of urease inhibitors is only reasonable in the presence of urea. The preliminary related investigations showed that the duration effect of the urease inhibitor D is dependent on temperature; no adverse effects caused by the inhibitor D of treated slurry on the biogas accumulation. Similar relations, as in cattle slurry, had been recognized and showed for the urease inhibitor D in pig slurry. As part of the investigations, a urease inhibitor is selected for cattle and pig slurry urea hydrolysis, and thus the ammonia release is significantly reduced. The basic investigations afforded an important contribution to the expansion of knowledge in this area, and lead on the other hand to develop new techniques in order to reduce the ammonia emissions from livestock housing.