Browsing by Subject "Chromatin"

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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.
Functional characterization of the COOH-terminal kinase activity of the TBP-associated factor TAF1
(2006) Maile, Tobias; Sauer, Frank
Activation of eukaryotic transcription involves an orchestrated interplay between transcription factors and the general RNA polymerase II (Pol II) transcription machinery (GTM), which consists of Pol II and general transcription factors (GTFs). The GTF TFIID consists of the TATA-box binding protein (TBP) and several TBP-associated factors (TAFs). The binding of TFIID to promoters can nucleate transcription. TAF1 is the largest subunit of TFIID and plays a central role within the nucleating function of TFIID in transcription. TAF1 mediates the binding of TFIID to promoters and interacts with enhancer-bound transcription factors and several GTFs. Additionally, TAF1 contains four enzymatic activities that are essential for viability of eukaryotes and mediate posttranslational modification of GTFs and histones. TAF1 is a bipartite protein kinase and contains an NH2-terminal kinase domain (NTK) and a COOH-terminal kinase domain (CTK). A previous study demonstrated that the CTK phosphorylates serine-residue 33 in histone H2B (H2BS33). However, the role of TAF1-mediated phosphorylation in transcription regulation remained unknown. In this study, the functional importance of H2BS33 phosphorylation (H2BS33P) by TAF1 was investigated by using a combination of biochemical and in vivo assays. In vitro kinase assays uncovered the two essential kinase motifs in TAF1CTK, the ATP-binding motif and the serine/threonine-specific catalytic motif, and indicate that the TAF1 CTK has intrinsic kinase-activity. Western blot analysis using an antibody to H2BS33P revealed that H2BS33 is phosphorylated in Drosophila. RNA-interference (RNAi) assays, designed to attenuate TAF1 expression (TAF1RNAi), revealed that TAF1 is a major kinase for H2BS33 in Drosophila Schneider cells. Flow-cytometry analysis of TAF1RNAi cells indicated that loss of TAF1 expression results in cell cycle arrest in G2/M-phase. Screening the transcription of cell cycle genes in TAF1RNAi cells by using reverse-transcriptase-PCR demonstrated that the transcription of the cell cycle gene string (stg) is reduced in the absence of TAF1. Chromatin immunoprecipitation assays (XChIP) indicate that H2BS33P is detectable at the transcriptionally active stg promoter but not at the silent stg promoter in TAF1RNAi cells. These results demonstrate that phosphorylation of H2BS33 is involved in stg transcription. XChIP-assays using chromatin prepared from Drosophila embryos, which express a mutant TAF1 lacking the CTK, revealed that CTK-mediated phosphorylation of H2BS33 plays an essential role in the activation of transcription of the Drosophila segmentation gene giant. In vitro kinase assays demonstrate that Bdf1 and Bdf2, the yeast homologues of the TAF1CTK, phosphorylate histones suggesting that the kinase activity of the TAF1CTK is phylogenetically conserved. The results of this work demonstrate that TAF1CTK is a major histone kinase of H2BS33 and that TAF1-mediated phosphorylation of H2BS33 plays an essential role in the transcription events during cell cycle progression and development.