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Dive into the research topics where Andrea Krempler is active.

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Featured researches published by Andrea Krempler.


Journal of Biological Chemistry | 2007

Ataxia Telangiectasia Mutated (ATM) Is Essential for DNA-PKcs Phosphorylations at the Thr-2609 Cluster upon DNA Double Strand Break

Benjamin P C Chen; Naoya Uematsu; Junya Kobayashi; Yaniv Lerenthal; Andrea Krempler; Hirohiko Yajima; Markus Löbrich; Yosef Shiloh; David J. Chen

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is rapidly phosphorylated at the Thr-2609 cluster and Ser-2056 upon ionizing radiation (IR). Furthermore, DNA-PKcs phosphorylation at both regions is critical for its role in DNA double strand break (DSB) repair as well as cellular resistance to radiation. IR-induced DNA-PKcs phosphorylation at Thr-2609 and Ser-2056, however, exhibits distinct kinetics indicating that they are differentially regulated. Although DNA-PKcs autophosphorylates itself at Ser-2056 after IR, we have reported here that ATM mediates DNA-PKcs phosphorylation at Thr-2609 as well as at the adjacent (S/T)Q motifs within the Thr-2609 cluster. In addition, our data suggest that DNA-PKcs- and ATM-mediated DNA-PKcs phosphorylations are cooperative and required for the full activation of DNA-PKcs and the subsequent DSB repair. Elimination of DNA-PKcs phosphorylation at both regions severely compromises radioresistance and DSB repair. Finally, our result provides a possible mechanism for the direct involvement of ATM in non-homologous end joining-mediated DSB repair.


Journal of Cell Biology | 2007

Chromosome breakage after G2 checkpoint release

Dorothee Deckbar; Julie Birraux; Andrea Krempler; Leopoldine Tchouandong; Andrea Beucher; Sarah Cusworth Walker; Tom Stiff; Penny A. Jeggo; Markus Löbrich

DNA double-strand break (DSB) repair and checkpoint control represent distinct mechanisms to reduce chromosomal instability. Ataxia telangiectasia (A-T) cells have checkpoint arrest and DSB repair defects. We examine the efficiency and interplay of ATMs G2 checkpoint and repair functions. Artemis cells manifest a repair defect identical and epistatic to A-T but show proficient checkpoint responses. Only a few G2 cells enter mitosis within 4 h after irradiation with 1 Gy but manifest multiple chromosome breaks. Most checkpoint-proficient cells arrest at the G2/M checkpoint, with the length of arrest being dependent on the repair capacity. Strikingly, cells released from checkpoint arrest display one to two chromosome breaks. This represents a major contribution to chromosome breakage. The presence of chromosome breaks in cells released from checkpoint arrest suggests that release occurs before the completion of DSB repair. Strikingly, we show that checkpoint release occurs at a point when approximately three to four premature chromosome condensation breaks and ∼20 γH2AX foci remain.


Molecular and Cellular Biology | 2004

Impaired Alveologenesis and Maintenance of Secretory Mammary Epithelial Cells in Jak2 Conditional Knockout Mice

Kay Uwe Wagner; Andrea Krempler; Aleata A. Triplett; Yongyue Qi; Nicholas M. George; Jianqiong Zhu; Hallgeir Rui

ABSTRACT Jak2 is a hormone-receptor-coupled kinase that mediates the tyrosine phosphorylation and activation of signal transducers and activators of transcription (Stat). The biological relevance of Jak2-Stat signaling in hormone-responsive adult tissues is difficult to investigate since Jak2 deficiency leads to embryonic lethality. We generated Jak2 conditional knockout mice to study essential functions of Jak2 during mammary gland development. The mouse mammary tumor virus-Cre-mediated excision of the first coding exon resulted in a Jak2 null mutation that uncouples signaling from the prolactin receptor (PRL-R) to its downstream mediator Stat5 in the presence of normal and supraphysiological levels of PRL. Jak2-deficient females were unable to lactate as a result of impaired alveologenesis. Unlike Stat5a knockouts, multiple gestation cycles could not reverse the Jak2-deficient phenotype, suggesting that neither other components of the PRL-R signaling cascade nor other growth factors and their signal transducers were able to compensate for the loss of Jak2 function to activate Stat5 in vivo. A comparative analysis of Jak2-deficient mammary glands with transplants from Stat5a/b knockouts revealed that Jak2 deficiency also impairs the pregnancy-induced branching morphogenesis. Jak2 conditional mutants therefore resemble PRL-R knockouts more closely, which suggested that Jak2 deficiency might affect additional PRL-R downstream mediators other than Stat5a and Stat5b. To address whether Jak2 is required for the maintenance of PRL-responsive, differentiating alveolar cells, we utilized a transgenic strain that expresses Cre recombinase under regulatory elements of the whey acidic protein gene (Wap). The Wap-Cre-mediated excision of Jak2 resulted in a negative selection of differentiated alveolar cells, suggesting that Jak2 is required not only for the proliferation and differentiation of alveolar cells but also for their maintenance during lactation.


Cell Cycle | 2007

An Imperfect G2M Checkpoint Contributes to Chromosome Instability Following Irradiation of S and G2 Phase Cells

Andrea Krempler; Dorothee Deckbar; Penny A. Jeggo; Markus Löbrich

DNA double strand break (DSB) repair and checkpoint control represent two major mechanisms that function to reduce chromosomal instability following ionising irradiation (IR). Ataxia telangiectasia (A-T) cells have long been known to have defective checkpoint responses. Recent studies have shown that they also have a DSB repair defect following IR raising the issue of how ATM’s repair and checkpoint functions interplay to maintain chromosomal stability. A-T and Artemis cells manifest an identical and epistatic repair defect throughout the cell cycle demonstrating that ATM’s major repair defect following IR represents Artemis-dependent end-processing. Artemis cells show efficient G2/M checkpoint induction and a prolonged arrest relative to normal cells. Following irradiation of G2 cells, this checkpoint is dependent on ATM and A-T cells fail to show checkpoint arrest. In contrast, cells irradiated during S phase initiate a G2/M checkpoint which is independent of ATM and, significantly, both Artemis and A-T cells show a prolonged arrest at the G2/M checkpoint likely reflecting their repair defect. Strikingly, the G2/M checkpoint is released before the completion of repair when approximately 10-20 DSBs remain both for S phase and G2 phase irradiated cells. This defined sensitivity level of the G2/M checkpoint explains the prolonged arrest in repair-deficient relative to normal cells and provides a conceptual framework for the co-operative phenotype between checkpoint and repair functions in maintaining chromosomal stability.


Journal of Biological Chemistry | 2004

Cell Cycle Arrest and Cell Death Are Controlled by p53-dependent and p53-independent Mechanisms in Tsg101-deficient Cells

Marissa J. Carstens; Andrea Krempler; Aleata A. Triplett; Maarten Van Lohuizen; Kay Uwe Wagner

Our previous studies have shown that cells conditionally deficient in Tsg101 arrested at the G1/S cell cycle checkpoint and died. We created a series of Tsg101 conditional knock-out cell lines that lack p53, p21Cip1, or p19Arf to determine the involvement of the Mdm2-p53 circuit as a regulator for G1/S progression and cell death. In this new report we show that the cell cycle arrest in Tsg101-deficient cells is p53-dependent, but a null mutation of the p53 gene is unable to maintain cell survival. The deletion of the Cdkn1a gene in Tsg101 conditional knock-out cells resulted in G1/S progression, suggesting that the p53-dependent G1 arrest in the Tsg101 knock-out is mediated by p21Cip1. The Cre-mediated excision of Tsg101 in immortalized fibroblasts that lack p19Arf seemed not to alter the ability of Mdm2 to sequester p53, and the p21-mediated G1 arrest was not restored. Based on these findings, we propose that the p21-dependent cell cycle arrest in Tsg101-deficient cells is an indirect consequence of cellular stress and not caused by a direct effect of Tsg101 on Mdm2 function as previously suggested. Finally, the deletion of Tsg101 from primary tumor cells that express mutant p53 and that lack p21Cip1 expression results in cell death, suggesting that additional transforming mutations during tumorigenesis do not affect the important role of Tsg101 for cell survival.


Radiation Research | 2008

Radiation-Induced HPRT Mutations Resulting from Misrejoined DNA Double-Strand Breaks

Kai Rothkamm; Kut Gunasekara; Salam A. Warda; Andrea Krempler; Markus Löbrich

Abstract Rothkamm, K., Gunasekara, K., Warda, S. A., Krempler, A. and Löbrich, M. Radiation-Induced HPRT Mutations Resulting from Misrejoined DNA Double-Strand Breaks. Radiat. Res. 169, 639–648 (2008). DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and α-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations.


Radiotherapy and Oncology | 2011

Elevated radiation-induced γH2AX foci in G2 phase heterozygous BRCA2 fibroblasts

Andrea Beucher; Dorothee Deckbar; Eik Schumann; Andrea Krempler; M. Frankenberg-Schwager; Markus Löbrich

BACKGROUND AND PURPOSE About 5-10% of all breast cancer cases are associated with heterozygous germ-line mutations in the genes encoding BRCA1 and BRCA2. Carriers of such mutations are highly predisposed for developing breast or ovarian cancer and, thus, are advised to undergo regular radio-diagnostic examinations. BRCA1 and BRCA2 are involved in multiple cellular processes including the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) and different studies addressing the DSB repair capacity of BRCA1+/- or BRCA2+/- cells led to contradictory results. MATERIALS AND METHODS Using the sensitive method of γH2AX foci analysis in combination with cell cycle markers, we specifically measured DSB repair in confluent G0 as well as in exponentially growing G1 and G2 phase primary WT, BRCA1+/- and BRCA2+/- fibroblasts. RESULTS Both BRCA1+/- and BRCA2+/- cells displayed normal DSB repair in G0 and in G1. In contrast, in G2, BRCA2+/- but not BRCA1+/- cells exhibited a decreased DSB repair capacity which was in between that of WT and that of a hypomorphic BRCA2-/- cell line. CONCLUSIONS The residual amount of normal BRCA1 seems to be sufficient for efficient DSB repair in all cell cycle phases, while the decreased DSB repair capacity of heterozygous BRCA2 mutations suggests gene dosage effects in G2.


International Journal of Oncology | 2013

Glioma-amplified sequence KUB3 influences double-strand break repair after ionizing radiation

Ulrike Fischer; Stefanie Rheinheimer; Andrea Krempler; Markus Löbrich; Eckart Meese

Human glioblastomas are characterized by frequent DNA amplifications most often at chromosome regions 7p11.2 and 12q13-15. Although amplification is a well-known hallmark of glioblastoma genetics the function of most amplified genes in glioblastoma biology is not understood. Previously, we cloned Ku70-binding protein 3 (KUB3) from the amplified domain at 12q13-15. Here, we report that glioblastoma cell cultures with endogenous KUB3 gene amplification and with elevated KUB3 protein expression show an efficient double-strand break (DSB) repair after being irradiated with 1 Gy. A significantly less efficient DSB repair was found in glioma cell cultures without KUB3 amplification and expression. Furthermore, we found that a siRNA-mediated reduction of the endogenous KUB3 expression in glioblastoma cells resulted in a reduction of the repair efficiency. HeLa cells transfected with KUB3 showed an increased DSB repair in comparison to untreated HeLa cells. In addition, KUB3 seems to influence DSB efficiency via the DNA-PK-dependent repair pathway as shown by simultaneous inhibition of KUB3 and DNA-PK. The data provide the first evidence for a link between the level of KUB3 amplification and expression in glioma and DSB repair efficiency.


Archive | 2005

Pathways of DNA Double-Strand Break Repair in Mammalian Cells after Ionizing Radiation

Andrea Krempler; Markus Löbrich

Exposure to ionizing radiation induces a considerable amount of double-strand breaks in mammalian DNA. Because this type of lesion substantially affects chromosomal integrity, double-strand breaks are considered as one of the most harmful DNA lesions. In order to avoid serious negative consequences for the cell and the whole organism, these breaks need to be reconstituted by highly efficient and precise repair mechanisms. Here, we review our current understanding of how the two main double-strand break repair mechanisms, non-homologous end joining and homologous recombination, operate to rejoin breaks induced by ionizing radiation.


Molecular Cell | 2004

A Pathway of Double-Strand Break Rejoining Dependent upon ATM, Artemis, and Proteins Locating to γ-H2AX Foci

Enriqueta Riballo; Martin Kühne; Nicole Rief; Aidan J. Doherty; Graeme Cameron Murray Smith; Marı́a-José Recio; Caroline Reis; Kirsten Dahm; Andreas Fricke; Andrea Krempler; Antony R. Parker; Andrew R. Gennery; Penny A. Jeggo; Markus Löbrich

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Markus Löbrich

Technische Universität Darmstadt

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Andrea Beucher

Technische Universität Darmstadt

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Dorothee Deckbar

Technische Universität Darmstadt

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Aleata A. Triplett

University of Nebraska Medical Center

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Kay Uwe Wagner

Eppley Institute for Research in Cancer and Allied Diseases

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Hallgeir Rui

Medical College of Wisconsin

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Jianqiong Zhu

Georgetown University Medical Center

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