Frank Wolf
Innsbruck Medical University
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Publication
Featured researches published by Frank Wolf.
The EMBO Journal | 2006
Frank Wolf; Cornelia Wandke; Nina Isenberg; Stephan Geley
The disassembly of the mitotic spindle and exit from mitosis require the inactivation of Cdk1. Here, we show that expression of nondegradable cyclinB1 causes dose‐dependent mitotic arrest phenotypes. By monitoring chromosomes in living cells, we determined that pronounced overexpression of stable cyclinB1 entailed metaphase arrest without detectable sister chromatid separation, while moderate overexpression arrested cells in a pseudometaphase state, in which separated sister chromatids were kept at the cellular equator by a bipolar ‘metaphase‐like’ spindle. Chromosomes that left the pseudometaphase plate became pulled back and individual kinetochores were found to be merotelically attached to both spindle poles in stable cyclinB1 arrested cells. Inactivation of the chromokinesin hKid, by RNAi or antibody microinjection, prevented the formation of stable bipolar spindles and the ‘metaphase‐like’ alignment of chromosomes in cells expressing stable cyclinB1. These experiments show that cyclinB1 is able to maintain a bipolar spindle even after sister chromatids had become separated and suggest an important role of hKid in this process. Cells expressing low levels of nondegradable cyclinB1 progressed further in mitosis and arrested in telophase.
Cell Stem Cell | 2010
Fang Li; Zhimin He; Jingping Shen; Qian Huang; Wenrong Li; Xinjian Liu; Yujun He; Frank Wolf; Chuan-Yuan Li
The molecular mechanisms involved in the derivation of induced pluripotent stem cells (iPSCs) from differentiated cells are poorly understood. Here we report that caspases 3 and 8, two proteases associated with apoptotic cell death, play critical roles in induction of iPSCs from human fibroblasts. Activation of caspases 3 and 8 occurs soon after transduction of iPSC-inducing transcription factors. Oct-4, a key iPSC transcription factor, is responsible for the activation. Inhibition of caspase 3 or 8 in human fibroblast cells partially or completely (respectively) prevents the induction of iPSCs. Furthermore, retinoblastoma susceptibility (Rb) protein appears to be one of the factors that act downstream of the caspases. We propose that caspases are key facilitators of nuclear reprogramming in iPSC induction.
Journal of Cell Biology | 2012
Cornelia Wandke; Marin Barisic; Reinhard Sigl; Veronika Rauch; Frank Wolf; Ana C. Amaro; Chia Huei Tan; António J. Pereira; Ulrike Kutay; Helder Maiato; Patrick Meraldi; Stephan Geley
Human chromokinesins hKID and KIF4A contribute to proper attachment of chromosomes by controlling the positioning of the chromosome arms and microtubule dynamics, respectively.
Cell Cycle | 2007
Frank Wolf; Reinhard Sigl; Stephan Geley
Exit from mitosis requires the proteolytic degradation of mitotic cyclins, which is instigated by the APC/C ubiquitin ligase. The coincidence of mitotic cyclin B1 degradation with the onset of anaphase intuitively suggested a requirement of cyclin degradation for sister chromatid separation. While this hypothesis has originally been refuted, evidence that cyclin B1 degradation is required for anaphase during meiosis has been obtained, while its requirement for anaphase during mitosis is still more controversial. By studying human cells engineered to express non-degradable cyclin B1, we have recently shown that stable cyclin B1 affects progression through mitosis at various steps in a dose-dependent manner. These experiments suggest that controlled exit from mitosis might involve CDK activity thresholds for important late mitotic events, such as the onset of anaphase, formation of the spindle midzone, the onset of cytokinesis, cellular abscission and chromosome decondensation.
Cancer Research | 2011
Wenrong Li; Fang Li; Qian Huang; Jingping Shen; Frank Wolf; Yujun He; Xinjian Liu; Y. Angela Hu; Joel S. Bedford; Chuan-Yuan Li
DNA double-strand breaks (DSB) are a major form of DNA damage and a key mechanism through which radiotherapy and some chemotherapeutic agents kill cancer cells. Despite its importance, measuring DNA DSBs is still a tedious task that is normally carried out by gel electrophoresis or immunofluorescence staining. Here, we report a novel approach to image and quantify DSBs in live mammalian cells through bifragment luciferase reconstitution. N- and C-terminal fragments of firefly luciferase genes were fused with H2AX and MDC1 genes, respectively. Our strategy was based on the established fact that at the sites of DSBs, H2AX protein is phosphoryated and physically associates with the MDC1 protein, thus bringing together N- and C-luciferase fragments and reconstituting luciferase activity. Our strategy allowed serial, noninvasive quantification of DSBs in cells irradiated with X-rays and (56)Fe ions. Furthermore, it allowed for the evaluation of DSBs noninvasively in vivo in irradiated tumors over 2 weeks. Surprisingly, we detected a second wave of DSB induction in irradiated tumor cells days after radiation exposure in addition to the initial rapid induction of DSBs. We conclude that our new split-luciferase-based method for imaging γ-H2AX-MDC1 interaction is a powerful new tool to study DSB repair kinetics in vivo with considerable advantage for experiments requiring observations over an extended period of time.
Investigative Ophthalmology & Visual Science | 2009
Xunda Ji; Lin Cheng; Fang Wei; Huiming Li; Mengyun Wang; Yuhua Tian; Xiafang Chen; Yufei Wang; Frank Wolf; Chuan-Yuan Li; Qian Huang
PURPOSE To establish human retinoblastoma (RB) animal models that allow sensitive, noninvasive and continuous monitoring of tumor growth and metastasis in vivo. METHODS The human RB tumor cell lines HXO-Rb44 and Y79 were engineered to express a fusion reporter gene allowing for bioluminescence and fluorescence imaging. Intraocular and metastatic tumors were induced in immunodeficient nude mice by injection of bioluminescent RB cells into eye compartments and into the left ventricle or tail vein. The growth kinetics of intraocular and metastatic tumors was quantitatively and continuously monitored via bioluminescence imaging (BLI). RESULTS Intraocular injection of HXO-Rb44-GFP-luc cells resulted in 100%, 80%, and 80% successful RB tumor development in the anterior chamber, vitreal cavity and subretinal space, respectively. The subretinal injection of Y79-GFP-luc cells resulted in 100% tumor development. BLI signal intensity correlated with the number of tumor cells injected as well as the weight of the tumor-bearing eyes. After bilateral subretinal injection of HXO-Rb44-GFP-luc cells, one of six RB tumor mice developed brain metastasis. Intracardiac injection of HXO-Rb44-GFP-luc cells resulted in metastatic disease in 9 of 15 nude mice, whereas tail vein injection resulted in metastasis in 1 of 16. Metastases were developed in multiple organs, including lymph nodes, bone, and brain, resembling the metastatic profile in patients with RB. CONCLUSIONS BLI allowed sensitive, noninvasive, and quantitative localization and monitoring of intraocular and metastatic RB tumor growth in vivo and thus may be a useful tool to study RB biology as well as anti-RB therapies.
Journal of Molecular Endocrinology | 2008
Karin Ecker; Andreas Lorenz; Frank Wolf; Christian Ploner; Günther Böck; Tod Duncan; Stephan Geley; Arno Helmberg
To search for proteins interacting with the glucocorticoid receptor, we adapted Aronheims reverse RAS recruitment system relying on the Saccharomyces cerevisiae mutant cdc25-2, which has a temperature-dependent defect in its RAS signaling pathway driving proliferation. The full-length human glucocorticoid receptor (NR3C1, isoform-alpha) was attached to the yeast plasma membrane in either of two orientations and used as bait to screen a HeLa cell cDNA library. Library proteins were fused to constitutively active, soluble human RAS, complementing the defective yeast pathway in case of bait-prey interaction. Screening of 800 000 clones resulted in the isolation of 21 proteins, 8 of which were followed up to evaluate interaction with the receptor in human cell lines. One of these candidates, the SCAN- and KRAB-domain-containing zinc finger protein 307 (ZKSCAN4) was co-precipitated with the receptor when both proteins were overexpressed in HEK293 cells. Rabbit antisera against ZKSCAN4 were raised, affinity purified, and used to immunoprecipitate endogenous ZKSCAN4 from Hct116 cells, resulting in co-precipitation of endogenous glucocorticoid receptor. Overexpressed ZKSCAN4 was found to co-localize in granular nuclear structures with the activated glucocorticoid receptor and partially with chromatin regions characterized by histone H3 mono-methylated on lysine 4 (H3K4me1). Overexpressed ZKSCAN4 had no effect on an episomal glucocorticoid receptor-driven reporter plasmid. By contrast, ZKSCAN4 markedly reduced glucocorticoid induction of the mouse mammary tumor virus-promoter-driven reporter gene when this was chromosomally integrated, arguing for a chromatin-dependent inhibition of glucocorticoid receptor-mediated transactivation.
Investigative Ophthalmology & Visual Science | 2012
Shenghai Zhang; Jihong Wu; Xiaobing Wu; Xu P; Yuhua Tian; Miaoying Yi; Xinjian Liu; Xiaoyan Dong; Frank Wolf; Chuan-Yuan Li; Qian Huang
PURPOSE Recombinant adeno-associated viral vector serotype 2 (rAAV2) has been used with success to deliver retina-targeted gene therapeutics in retinal degeneration. However, one of the major limitations of this approach is the vectors low transduction efficiency. This study is designed to increase AAV2 transduction efficiency in vitro and in vivo. METHODS Green fluorescence protein (GFP) or luciferase reporter gene-carried rAAV2 vectors were applied to cultured human RPE cells (ARPE-19) or animal eyes with or without chemotherapeutic agents. GFP transduction efficiency was evaluated by image, flow cytometry analysis, and Western blot. The ciliary neurotrophic factor (rAAV2-CNTF)-carried AAV2 vector was coinjected to subretinal space with or without chemotherapeutic agent. The therapeutic efficacy was evaluated by counting numbers of remaining photoreceptors in retina sections of treated or untreated eyes. RESULTS Coadministration of 0.1 μg/mL doxorubicin (DXR), 0.14 μg/mL cytarabine (Ara-C), 1 μg/mL etoposide (VP-16), or 20 μg/mL cisplatin (DDP) significantly increased rAAV2-mediated GFP and/or luciferase expression in cultured hRPE cells without any detectable toxicity. Pretreatment with DXR for 24 h prior to infection was most effective in enhancing rAAV2 transgene expression in hRPE cells. In addition, subretinal coinjection of rAAV2-CMV-ciliary neurotrophic factor (rAAV2-CNTF) and DXR into the eyes of rats with inherited retinal degeneration resulted in an approximately 2-fold increase in photoreceptor layer thickness and cellular density of the outer nuclear layer (ONL) compared to rAAV2-CNTF alone, reflecting a pronounced protection effect mediated by the enhanced expression of CNTF. CONCLUSIONS The method described here to improve rAAV2-based gene delivery is simple and feasible without any detectable toxicity. This strategy might be therapeutically exploited in the gene therapy of degenerative retinal diseases.
International Journal of Radiation Oncology Biology Physics | 2011
Frank Wolf; Wenrong Li; Fang Li; Chuan-Yuan Li
PURPOSE To develop a split-luciferase-based reporter system that allows for noninvasive monitoring of activation of the Her2/neu pathway in vivo in a quantitative and sensitive manner. METHODS AND MATERIALS Fusion proteins of the ErbB2/Her2/neu receptor to the N-terminal fragment of luciferase and of its downstream binding partner Shc to the C-terminal fragment of luciferase have been engineered owing to the rationale that on activation and binding of the Her2 receptor molecule to Shc, luciferase function will be reconstituted. Thus, the resulting bioluminescence signals can serve as a surrogate measure of receptor activation. RESULTS We have shown that our reporter systems functions well in vitro in breast cancer cells and in vivo in xenograft tumors. In particular, the activities of Her2/neu in xenograft tumors could be monitored serially for an extended period after radiotherapy. CONCLUSIONS We believe that the novel ErbB2/Her2/neu reporter we have presented is a powerful tool to study the biology of the Her2-neu pathway in vitro and in vivo. It should also facilitate the development and rapid evaluation of new Her2/neu-targeted therapeutic agents.
Human Gene Therapy | 2010
Xinjian Liu; Jinjun Li; Yuhua Tian; Xu P; Xiafang Chen; Kuangchen Xie; Zhengjun Qiu; Yufei Wang; Dabing Zhang; Frank Wolf; Chuan-Yuan Li; Qian Huang
Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, and even under optimal therapy these patients face a poor prognosis. Here we report a novel gene therapy-based strategy to battle this disease. We show that the majority of pancreatic tumors overexpress c-erb-B2, which therefore might serve as a target for novel therapies. On the basis of these findings, we developed an adenoviral vector [Ad-e23(scFv)-PE40] encoding a c-erb-B2 (Her-2/neu)-targeted immunotoxin. To improve viral gene delivery we coinfected the therapeutic adenovirus with a replication-competent adenovirus (RCAd) at low doses that enhanced the transduction efficiency of the former virus. In addition, we show that target gene expression can be enhanced by adding etoposide (VP16) at nontherapeutic doses. To investigate the therapeutic efficacy of our approach we established a mouse model for advanced pancreatic cancer disease by intraperitoneal injection of pancreatic cancer cell lines, resulting in multifocal peritoneal xenograft tumors. Administration of Ad-e23(scFv)-PE40 in combination with RCAd and VP16 significantly inhibited tumor growth in mice, with no apparent systemic toxicity. In this study we show that c-erb-B2 might be an effective molecular target in the treatment of pancreatic tumors and that coadministration of a therapeutic c-erb-B2-targeted, non-replication-competent adenovirus with an RCAd and VP16 could be a powerful approach to effectively deliver therapeutic genes to tumors. As demonstrated, this strategy can be employed to effectively treat pancreatic cancer in particular, but may be modified to treat other types of cancer as well.