DOCSLIB.ORG

Gene and Protein Expression Profiling of Human Ovarian Cancer Cells Treated with the Heat Shock Protein 90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Gene and Protein Expression Profiling of Human Ovarian Cancer Cells Treated with the Heat Shock Protein 90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin Research Article Gene and Protein Expression Profiling of Human Ovarian Cancer Cells Treated with the Heat Shock Protein 90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin Alison Maloney,1 Paul A. Clarke,1 Soren Naaby-Hansen,3,4 Rob Stein,3,5 Jens-Oliver Koopman,3,4 Akunna Akpan,3,4 Alice Yang,3,4 Marketa Zvelebil,3,4 Rainer Cramer,3,4 Lindsay Stimson,1 Wynne Aherne,1 Udai Banerji,1,2 Ian Judson,1,2 Swee Sharp,1 Marissa Powers,1 Emmanuel deBilly,1 Joanne Salmons,1 Michael Walton,1 Al Burlingame,3,4 Michael Waterfield,3,4 and Paul Workman1 1Haddow Laboratories, Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research; 2Royal Marsden NHS Foundation Trust, Sutton, Surrey, United Kingdom; 3Ludwig Institute for Cancer Research and Departments of 4Biochemistry and Molecular Biology and 5Oncology, University College London, London, United Kingdom Abstract anticancer agents and provide a means of obtaining a detailed The promising antitumor activity of 17-allylamino-17-deme- molecular signature of drug action (1, 2). In addition, these thoxygeldanamycin (17AAG) results from inhibition of the methods may identify pharmacodynamic markers that can be used molecular chaperone heat shock protein 90(HSP90)and to evaluate drugs in clinical trials. Gene expression microarrays are subsequent degradation of multiple oncogenic client proteins. increasingly used to investigate the molecular responses to cancer Gene expression microarray and proteomic analysis were used drugs in tumor cells (1). Although valuable, analysis of gene to profile molecular changes in the A2780human ovarian expression at the mRNA level alone cannot adequately predict cancer cell line treated with 17AAG. Comparison of results protein expression or functional states. Therefore, proteomic with an inactive analogue and an alternative HSP90inhibitor approaches also have considerable potential in the drug develop- radicicol indicated that increased expression of HSP72, ment process (2). Heat shock proteins (HSPs) are a major class of molecular HSC70, HSP27, HSP47, and HSP90B at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were chaperones that play a vital role in the cellular stress response increased in tumor biopsies following treatment of patients and cancer (3, 4). One particular chaperone, HSP90, is of with 17AAG. A group of MYC-regulated mRNAs was decreased considerable current interest as a new cancer therapeutic target by 17AAG. Of particular interest and novelty were changes because of its essential role in maintaining the conformational in expression of chromatin-associated proteins. Expression of stability and function of a number of oncogenic ‘‘client’’ proteins the heterochromatin protein 1 was increased, and expression that are required for cellular proliferation, cell cycle regulation, of the histone acetyltransferase 1 and the histone arginine apoptosis, invasion, angiogenesis, and metastasis (for review, see methyltransferase PRMT5 was decreased by 17AAG. PRMT5 refs. 4, 5). The natural product HSP90 inhibitors radicicol, was shown to be a novel HSP90-binding partner and potential geldanamycin, and their derivatives exert their antitumor effect by inhibiting the intrinsic ATPase activity of HSP90, resulting in client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction degradation of HSP90 client proteins via the ubiquitin-protea- on cell proliferation with the histone deacetylase inhibitor some pathway (4, 5). Based on its novel mechanism of action trichostatin A. This mRNA and protein expression analysis has and the potential for combinatorial effects on multiple provided new insights into the complex molecular pharma- oncogenic pathways and on all of the hallmarks of cancer (6), cology of 17AAG and suggested new genes and proteins that together with promising therapeutic effects in animal models may be involved in response to the drug or be potential (e.g., refs. 7–9), the geldanamycin analogue 17-allylamino-17- biomarkers of drug action. [Cancer Res 2007;67(7):3239–53] demethoxygeldanamycin (17AAG), a benzoquinone ansamycin, has completed phase I and is now in phase II clinical trials as the first-in-class HSP90 inhibitor (10, 11). Other HSP90 inhibitors Introduction are also in development (5). Gene expression microarray and proteomic profiling facilitate Gene expression profiling studies have been done previously in screening for the response of thousands of mRNAs and proteins to a panel of human colon cancer cell lines following treatment with 17AAG (12). That study identified HSC70 (HSPA8) and HSP90b (HSPCB) as 17AAG-responsive genes (12). Alongside depletion in Note: Supplementary data for this article are available at Cancer Research Online levels of client proteins, such as c-RAF-1 and cyclin-dependent (http://cancerres.aacrjournals.org/). kinase 4 (CDK4), inducible HSP72 (HSPA1A/HSPA1B) has been P. Workman is a Cancer Research UK Life Fellow. Conflict of interest: P. Workman’s laboratory has received support from Vernalis, used as a pharmacodynamic end point in phase I clinical trials of and HSP90 inhibitor technology from The Institute of Cancer Research/Vernalis 17AAG (e.g., ref. 10). Indeed, this basic molecular signature has collaboration has been licensed to Novartis. Requests for reprints: Paul Workman, Haddow Laboratories, Cancer Research been used to show HSP90 inhibition in the tumor tissue of UK Centre for Cancer Therapeutics, 15 Cotswold Road, Belmont, Sutton, Surrey SM2 treated patients, and this was associated with prolonged stable 5NG, United Kingdom. Phone: 44-20-8722-4301; Fax: 44-20-8722-4324; E-mail: paul. disease in two patients with metastatic malignant melanoma (10). [email protected]. I2007 American Association for Cancer Research. The cellular response to HSP90 inhibition has a complex nature doi:10.1158/0008-5472.CAN-06-2968 that involves both multiple protein changes and effects on www.aacrjournals.org 3239 Cancer Res 2007; 67: (7). April 1, 2007 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Cancer Research transcription. We hypothesized that by using proteomic analysis obtained from Sigma Chemical Co. (Poole, United Kingdom). Compounds in combination with gene expression microarray profiling we were stored as 2 mmol/L stocks in DMSO at À20jC and protected from might identify additional molecular responses to 17AAG that light. All reagents were from Sigma unless stated otherwise and were of would increase our understanding of the mechanism of action of highest chemical grade. the drug. This approach also has the potential to facilitate the Combination treatments. A2780 cells were seeded at 1,000 per well in 96-well plates. Sulfurhodamine blue staining was used to determine the IC discovery of new HSP90 client proteins as well as to suggest novel 50 at 96 h for TSA and 17AAG. These values were then used in combination pharmacodynamic markers. studies according to the median effect analysis method of Chou and Talalay We now report a study in which the complementary (13). Briefly, increasing concentrations of TSA and 17AAG were added at a approaches of mRNA and protein expression profiling have been ratio of 1:1 based on their respective IC50 values. Following a 96-h exposure, used in parallel to examine expression changes in response to cells were stained with sulfurhodamine blue, and a combination index for drug treatment in a human cancer cell line. Changes in mRNA nonexclusive interactions was calculated using an algorithm based on that and protein expression were determined following treatment of described by Chou and Talalay (13). A combination index of 1 indicates an a human ovarian adenocarcinoma cell line with 17AAG. In additive interaction; a combination index < 1 indicates a synergistic addition, we sought to distinguish ‘‘on-target’’ effects (those which interaction; and a combination index > 1 indicates an antagonistic are a consequence of HSP90 inhibition) from ‘‘off-target’’ effects interaction. Protein extraction. A2780 cells were plated in 75-cm2 dishes at a (those which relate to other interactions) by comparison of the density of 1 Â 106 and left to attach for 24 h. Control cells were set up in molecular responses obtained following treatment with 17AAG quadruplicate, and 17AAG-treated cells set up in duplicate. Additional with those seen with the chemically dissimilar HSP90 inhibitor dishes were set up for cDNA microarray and Western blotting analysis. radicicol or an inactive analogue of 17AAG. Selected known and Cells were treated for 24 h with pharmacologically relevant, isoeffective novel expression changes were followed up by validation using concentrations of 17AAG (60 nmol/L) and radicicol (600 nmol/L), or, in Western blotting and investigation of functional and therapeutic some experiments, with an equimolar concentration of the inactive significance. 17AAG analogue and radicicol in relation to 17AAG (60 nmol/L) in 10 mL Expression profiling studies revealed that around 3% of the medium. total gene transcripts examined and 4% of the detectable proteins Cell lysates for proteomic analysis were prepared in a laminar flow were responsive to 17AAG treatment. The changes seen involved hood by scraping the cells into lysis buffer containing 8 mol/L urea (Merck, Poole, United Kingdom), 2 mol/L thiourea (Merck), 4% CHAPS, 65 a number of molecular chaperones, protein synthesis and degra- mmol/L DTT (Merck), and a protease
Load more

Recommended publications
  • Lupus-Associated Endogenous Retroviral LTR Polymorphism and Epigenetic Imprinting Promote HRES-1/Rab4 Expression and Mtor Activation
    Lupus-associated endogenous retroviral LTR polymorphism and epigenetic imprinting promote HRES-1/Rab4 expression and mTOR activation Aparna Godavarthy, … , Katalin Banki, Andras Perl JCI Insight. 2019. https://doi.org/10.1172/jci.insight.134010. Research In-Press Preview Immunology Graphical abstract Find the latest version: https://jci.me/134010/pdf LUPUS-ASSOCIATED ENDOGENOUS RETROVIRAL LTR POLYMORPHISM AND EPIGENETIC IMPRINTING PROMOTE HRES-1/RAB4 EXPRESSION AND MTOR ACTIVATION Aparna Godavarthy*1, Ryan Kelly*1, John Jimah1, Miguel Beckford1, Tiffany Caza1,2, David Fernandez1,2, Nick Huang1,3, Manuel Duarte1,2, Joshua Lewis1,2, Hind J. Fadel4, Eric M. Poeschla4, Katalin Banki5, and Andras Perl1,2,3 * These authors contributed equally to the study. 1, Division of Rheumatology, Department of Medicine; 2, Department of Microbiology and Immunology; 3, Department of Biochemistry and Molecular Biology, and 5, Department of Pathology, State University of New York, Upstate Medical University, College of Medicine, 750 East Adams Street, Syracuse, New York 13210; 4, Department of Molecular Medicine; Mayo Clinic College of Medicine, 200 First Street SW, Rochester 55905, USA; Correspondence: Andras Perl, M.D., Ph.D. , State University of New York, College of Medicine, 750 East Adams Street, Syracuse, New York 13210; Phone: (315) 464-4194; Fax: (315) 464- 4176; E-mail: [email protected] Key Words: Systemic Lupus Erythematosus, T Cells, HRES-1/Rab4. mTOR, Autoimmunity The authors have declared that no conflict of interest exists. Supplementary Materials include Supplemental Methods and Supplementary Figures S1-S26. 1 ABSTRACT Overexpression and long terminal repeat (LTR) polymorphism of the HRES-1/Rab4 human endogenous retrovirus locus have been associated with T-cell activation and disease manifestations in systemic lupus erythematosus (SLE).
    [Show full text]
  • Topoisomerase 1 Suppresses Replication Stress and Genomic Instability by Preventing Interference Between Replication and Transcription
    Topoisomerase 1 suppresses replication stress and genomic instability by preventing interference between replication and transcription. Sandie Tuduri, Laure Crabbé, Chiara Conti, Hélène Tourrière, Heidi Holtgreve-Grez, Anna Jauch, Véronique Pantesco, John de Vos, Aubin Thomas, Charles Theillet, et al. To cite this version: Sandie Tuduri, Laure Crabbé, Chiara Conti, Hélène Tourrière, Heidi Holtgreve-Grez, et al.. Topoi- somerase 1 suppresses replication stress and genomic instability by preventing interference between replication and transcription.. Nature Cell Biology, Nature Publishing Group, 2009, 11 (11), pp.1315- 1324. 10.1038/ncb1984. hal-00430775 HAL Id: hal-00430775 https://hal.archives-ouvertes.fr/hal-00430775 Submitted on 14 Jun 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Topoisomerase I suppresses genomic instability by preventing interference between replication and transcription Sandie Tuduri 1,2, Laure Crabbé 1, Chiara Conti 3, Hélène Tourrière 1, Heidi Holtgreve-Grez 4, Anna Jauch 4, Véronique Pantesco 5, John De Vos 5, Aubin
    [Show full text]
  • 1 Supporting Information for a Microrna Network Regulates
    Supporting Information for A microRNA Network Regulates Expression and Biosynthesis of CFTR and CFTR-ΔF508 Shyam Ramachandrana,b, Philip H. Karpc, Peng Jiangc, Lynda S. Ostedgaardc, Amy E. Walza, John T. Fishere, Shaf Keshavjeeh, Kim A. Lennoxi, Ashley M. Jacobii, Scott D. Rosei, Mark A. Behlkei, Michael J. Welshb,c,d,g, Yi Xingb,c,f, Paul B. McCray Jr.a,b,c Author Affiliations: Department of Pediatricsa, Interdisciplinary Program in Geneticsb, Departments of Internal Medicinec, Molecular Physiology and Biophysicsd, Anatomy and Cell Biologye, Biomedical Engineeringf, Howard Hughes Medical Instituteg, Carver College of Medicine, University of Iowa, Iowa City, IA-52242 Division of Thoracic Surgeryh, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Canada-M5G 2C4 Integrated DNA Technologiesi, Coralville, IA-52241 To whom correspondence should be addressed: Email: [email protected] (M.J.W.); yi- [email protected] (Y.X.); Email: [email protected] (P.B.M.) This PDF file includes: Materials and Methods References Fig. S1. miR-138 regulates SIN3A in a dose-dependent and site-specific manner. Fig. S2. miR-138 regulates endogenous SIN3A protein expression. Fig. S3. miR-138 regulates endogenous CFTR protein expression in Calu-3 cells. Fig. S4. miR-138 regulates endogenous CFTR protein expression in primary human airway epithelia. Fig. S5. miR-138 regulates CFTR expression in HeLa cells. Fig. S6. miR-138 regulates CFTR expression in HEK293T cells. Fig. S7. HeLa cells exhibit CFTR channel activity. Fig. S8. miR-138 improves CFTR processing. Fig. S9. miR-138 improves CFTR-ΔF508 processing. Fig. S10. SIN3A inhibition yields partial rescue of Cl- transport in CF epithelia.
    [Show full text]
  • Aneuploidy: Using Genetic Instability to Preserve a Haploid Genome?
    Health Science Campus FINAL APPROVAL OF DISSERTATION Doctor of Philosophy in Biomedical Science (Cancer Biology) Aneuploidy: Using genetic instability to preserve a haploid genome? Submitted by: Ramona Ramdath In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Science Examination Committee Signature/Date Major Advisor: David Allison, M.D., Ph.D. Academic James Trempe, Ph.D. Advisory Committee: David Giovanucci, Ph.D. Randall Ruch, Ph.D. Ronald Mellgren, Ph.D. Senior Associate Dean College of Graduate Studies Michael S. Bisesi, Ph.D. Date of Defense: April 10, 2009 Aneuploidy: Using genetic instability to preserve a haploid genome? Ramona Ramdath University of Toledo, Health Science Campus 2009 Dedication I dedicate this dissertation to my grandfather who died of lung cancer two years ago, but who always instilled in us the value and importance of education. And to my mom and sister, both of whom have been pillars of support and stimulating conversations. To my sister, Rehanna, especially- I hope this inspires you to achieve all that you want to in life, academically and otherwise. ii Acknowledgements As we go through these academic journeys, there are so many along the way that make an impact not only on our work, but on our lives as well, and I would like to say a heartfelt thank you to all of those people: My Committee members- Dr. James Trempe, Dr. David Giovanucchi, Dr. Ronald Mellgren and Dr. Randall Ruch for their guidance, suggestions, support and confidence in me. My major advisor- Dr. David Allison, for his constructive criticism and positive reinforcement.
    [Show full text]
  • Early Growth Response 1 Regulates Hematopoietic Support and Proliferation in Human Primary Bone Marrow Stromal Cells
    Hematopoiesis SUPPLEMENTARY APPENDIX Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells Hongzhe Li, 1,2 Hooi-Ching Lim, 1,2 Dimitra Zacharaki, 1,2 Xiaojie Xian, 2,3 Keane J.G. Kenswil, 4 Sandro Bräunig, 1,2 Marc H.G.P. Raaijmakers, 4 Niels-Bjarne Woods, 2,3 Jenny Hansson, 1,2 and Stefan Scheding 1,2,5 1Division of Molecular Hematology, Department of Laboratory Medicine, Lund University, Lund, Sweden; 2Lund Stem Cell Center, Depart - ment of Laboratory Medicine, Lund University, Lund, Sweden; 3Division of Molecular Medicine and Gene Therapy, Department of Labora - tory Medicine, Lund University, Lund, Sweden; 4Department of Hematology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands and 5Department of Hematology, Skåne University Hospital Lund, Skåne, Sweden ©2020 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2019.216648 Received: January 14, 2019. Accepted: July 19, 2019. Pre-published: August 1, 2019. Correspondence: STEFAN SCHEDING - [email protected] Li et al.: Supplemental data 1. Supplemental Materials and Methods BM-MNC isolation Bone marrow mononuclear cells (BM-MNC) from BM aspiration samples were isolated by density gradient centrifugation (LSM 1077 Lymphocyte, PAA, Pasching, Austria) either with or without prior incubation with RosetteSep Human Mesenchymal Stem Cell Enrichment Cocktail (STEMCELL Technologies, Vancouver, Canada) for lineage depletion (CD3, CD14, CD19, CD38, CD66b, glycophorin A). BM-MNCs from fetal long bones and adult hip bones were isolated as reported previously 1 by gently crushing bones (femora, tibiae, fibulae, humeri, radii and ulna) in PBS+0.5% FCS subsequent passing of the cell suspension through a 40-µm filter.
    [Show full text]
  • Heat Shock Proteins in Vascular Diabetic Complications: Review and Future Perspective
    International Journal of Molecular Sciences Review Heat Shock Proteins in Vascular Diabetic Complications: Review and Future Perspective Stefania Bellini 1,†, Federica Barutta 1,†, Raffaella Mastrocola 2 ID , Luigi Imperatore 1, Graziella Bruno 1 and Gabriella Gruden 1,* 1 Laboratory of Diabetic Nephropathy, Department of Medical Sciences, University of Turin, Corso Dogliotti 14, 10126 Turin, Italy; [email protected] (S.B.); [email protected] (F.B.); [email protected] (L.I.); [email protected] (G.B.) 2 Department of Clinical and Biological Sciences, University of Turin, Corso Raffaello 30, 10125 Turin, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-011-633-6035 † These authors contribute equally to this work. Received: 27 November 2017; Accepted: 11 December 2017; Published: 14 December 2017 Abstract: Heat shock proteins (HSPs) are a large family of proteins highly conserved throughout evolution because of their unique cytoprotective properties. Besides assisting protein refolding and regulating proteostasis under stressful conditions, HSPs also play an important role in protecting cells from oxidative stress, inflammation, and apoptosis. Therefore, HSPs are crucial in counteracting the deleterious effects of hyperglycemia in target organs of diabetes vascular complications. Changes in HSP expression have been demonstrated in diabetic complications and functionally related to hyperglycemia-induced cell injury. Moreover, associations between diabetic complications and altered circulating levels of both HSPs and anti-HSPs have been shown in clinical studies. HSPs thus represent an exciting therapeutic opportunity and might also be valuable as clinical biomarkers. However, this field of research is still in its infancy and further studies in both experimental diabetes and humans are required to gain a full understanding of HSP relevance.
    [Show full text]
  • Figure S1. HAEC ROS Production and ML090 NOX5-Inhibition
    Figure S1. HAEC ROS production and ML090 NOX5-inhibition. (a) Extracellular H2O2 production in HAEC treated with ML090 at different concentrations and 24 h after being infected with GFP and NOX5-β adenoviruses (MOI 100). **p< 0.01, and ****p< 0.0001 vs control NOX5-β-infected cells (ML090, 0 nM). Results expressed as mean ± SEM. Fold increase vs GFP-infected cells with 0 nM of ML090. n= 6. (b) NOX5-β overexpression and DHE oxidation in HAEC. Representative images from three experiments are shown. Intracellular superoxide anion production of HAEC 24 h after infection with GFP and NOX5-β adenoviruses at different MOIs treated or not with ML090 (10 nM). MOI: Multiplicity of infection. Figure S2. Ontology analysis of HAEC infected with NOX5-β. Ontology analysis shows that the response to unfolded protein is the most relevant. Figure S3. UPR mRNA expression in heart of infarcted transgenic mice. n= 12-13. Results expressed as mean ± SEM. Table S1: Altered gene expression due to NOX5-β expression at 12 h (bold, highlighted in yellow). N12hvsG12h N18hvsG18h N24hvsG24h GeneName GeneDescription TranscriptID logFC p-value logFC p-value logFC p-value family with sequence similarity NM_052966 1.45 1.20E-17 2.44 3.27E-19 2.96 6.24E-21 FAM129A 129. member A DnaJ (Hsp40) homolog. NM_001130182 2.19 9.83E-20 2.94 2.90E-19 3.01 1.68E-19 DNAJA4 subfamily A. member 4 phorbol-12-myristate-13-acetate- NM_021127 0.93 1.84E-12 2.41 1.32E-17 2.69 1.43E-18 PMAIP1 induced protein 1 E2F7 E2F transcription factor 7 NM_203394 0.71 8.35E-11 2.20 2.21E-17 2.48 1.84E-18 DnaJ (Hsp40) homolog.
    [Show full text]
  • Regulation of Heterochromatic Gene Silencing in Mouse
    Aus dem Adolf-Butenandt-Institut der Ludwig-Maximilians-Universität München Lehrstuhl: Molekularbiologie Direktor: Prof. Dr. Peter B. Becker Arbeitsgruppe: Prof. Dr. Gunnar Schotta Regulation of heterochromatic gene silencing in mouse Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) an der Medizinischen Fakultät der Ludwig-Maximilians-Universität München vorgelegt von Dennis Šadić aus Daun München, 2014 2 Gedruckt mit Genehmigung der Medizinischen Fakultät der Ludwig-Maximilians- Universität München Betreuer: Prof. Dr. Gunnar Schotta Zweitgutachter: Prof. Dr. Heiko Hermeking Dekan: Prof. Dr. Dr. h.c. Maximilian Reiser, FACR, FRCR Tag der mündlichen Prüfung: 19.09.2014 3 Eidesstattliche Versicherung Ich erkläre hiermit an Eides statt, dass ich die vorliegende Dissertation mit dem Thema “Regulation of heterochromatic gene silencing in mouse” selbständig verfasst, mich außer der angegebenen keiner weiteren Hilfsmittel bedient und alle Erkenntnisse, die aus dem Schrifttum ganz oder annähernd übernommen sind, als solche kenntlich gemacht und nach ihrer Herkunft unter Bezeichnung der Fundstelle einzeln nachgewiesen habe. Ich erkläre des Weiteren, dass die hier vorgelegte Dissertation nicht in gleicher oder in ähnlicher Form bei einer anderen Stelle zur Erlangung eines akademischen Grades eingereicht wurde. ____________________ ______________________________________ Ort, Datum Unterschrift Dennis Šadić 4 The work of my thesis is under preparation for a publication in a peer-reviewed journal. During my PhD thesis I collaborated with others in the following scientific projects: Dambacher, S., Deng, W., Hahn, M., Sadic, D., Frohlich, J., Nuber, A., Hoischen, C., Diekmann, S., Leonhardt, H., and Schotta, G. (2012). CENP-C facilitates the recruitment of M18BP1 to centromeric chromatin. Nucleus 3, 101-110. Hahn, M., Dambacher, S., Dulev, S., Kuznetsova, A.Y., Eck, S., Worz, S., Sadic, D., Schulte, M., Mallm, J.P., Maiser, A., et al.
    [Show full text]
  • Transcriptional Recapitulation and Subversion Of
    Open Access Research2007KaiseretVolume al. 8, Issue 7, Article R131 Transcriptional recapitulation and subversion of embryonic colon comment development by mouse colon tumor models and human colon cancer Sergio Kaiser¤*, Young-Kyu Park¤†, Jeffrey L Franklin†, Richard B Halberg‡, Ming Yu§, Walter J Jessen*, Johannes Freudenberg*, Xiaodi Chen‡, Kevin Haigis¶, Anil G Jegga*, Sue Kong*, Bhuvaneswari Sakthivel*, Huan Xu*, Timothy Reichling¥, Mohammad Azhar#, Gregory P Boivin**, reviews Reade B Roberts§, Anika C Bissahoyo§, Fausto Gonzales††, Greg C Bloom††, Steven Eschrich††, Scott L Carter‡‡, Jeremy E Aronow*, John Kleimeyer*, Michael Kleimeyer*, Vivek Ramaswamy*, Stephen H Settle†, Braden Boone†, Shawn Levy†, Jonathan M Graff§§, Thomas Doetschman#, Joanna Groden¥, William F Dove‡, David W Threadgill§, Timothy J Yeatman††, reports Robert J Coffey Jr† and Bruce J Aronow* Addresses: *Biomedical Informatics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA. †Departments of Medicine, and Cell and Developmental Biology, Vanderbilt University and Department of Veterans Affairs Medical Center, Nashville, TN 37232, USA. ‡McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706, USA. §Department of Genetics and Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599, USA. ¶Molecular Pathology Unit and Center for Cancer Research, Massachusetts deposited research General Hospital, Charlestown, MA 02129, USA. ¥Division of Human Cancer Genetics, The Ohio State University College of Medicine, Columbus, Ohio 43210-2207, USA. #Institute for Collaborative BioResearch, University of Arizona, Tucson, AZ 85721-0036, USA. **University of Cincinnati, Department of Pathology and Laboratory Medicine, Cincinnati, OH 45267, USA. ††H Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA. ‡‡Children's Hospital Informatics Program at the Harvard-MIT Division of Health Sciences and Technology (CHIP@HST), Harvard Medical School, Boston, Massachusetts 02115, USA.
    [Show full text]
  • Investigation Into the Structure and Function of Hsp47
    Investigation into the structure and function of Hsp47 Umbreen Ahmed A thesis submitted to the University of Birmingham for the degree of Doctor of Philosophy School of Biosciences University of Birmingham Birmingham B15 2TT May 2009 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Acknowledgements Firstly, a huge thanks to my supervisor, Dr Timothy Dafforn for his continuous support, enthusiasm and patience throughout my PhD. It has been a pleasure working for him, even when the lab work has not been particularly rewarding! Our many conversations on cars and formula 1 have been especially enjoyable and will be missed. I would also like to thank our collaborators from Imperial College (Andrew Miller, Dee Olerenshaw, Takayuki Homma, Micheal Wright and Firdaus Abdul) who provided me with the Mus musculus Hsp47 clones. Their mutagenesis work has made this thesis possible and our fruitful discussions have been both interesting and helpful. Thanks also to the James Whisstock group at Monash University, Australia, for providing me with the opportunity to work with such enthusiastic and dedicated scientists. I am indebted to others for their valuable time, expertise and use of instrumentation with regard to specific techniques; Dr Corrine Smith for electron microscopy and Dr Klaus Fütterer for protein crystallography.
    [Show full text]
  • Ultraconserved Elements Are Associated with Homeostatic Control of Splicing Regulators by Alternative Splicing and Nonsense-Mediated Decay
    Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay Julie Z. Ni,1 Leslie Grate,1 John Paul Donohue,1 Christine Preston,2 Naomi Nobida,2 Georgeann O’Brien,2 Lily Shiue,1 Tyson A. Clark,3 John E. Blume,3 and Manuel Ares Jr.1,2,4 1Center for Molecular Biology of RNA and Department of Molecular, Cell, and Developmental Biology, University of California at Santa Cruz, Santa Cruz, California 95064, USA; 2Hughes Undergraduate Research Laboratory, University of California at Santa Cruz, Santa Cruz, California 95064, USA; 3Affymetrix, Inc., Santa Clara, California 95051, USA Many alternative splicing events create RNAs with premature stop codons, suggesting that alternative splicing coupled with nonsense-mediated decay (AS-NMD) may regulate gene expression post-transcriptionally. We tested this idea in mice by blocking NMD and measuring changes in isoform representation using splicing-sensitive microarrays. We found a striking class of highly conserved stop codon-containing exons whose inclusion renders the transcript sensitive to NMD. A genomic search for additional examples identified >50 such exons in genes with a variety of functions. These exons are unusually frequent in genes that encode splicing activators and are unexpectedly enriched in the so-called “ultraconserved” elements in the mammalian lineage. Further analysis show that NMD of mRNAs for splicing activators such as SR proteins is triggered by splicing activation events, whereas NMD of the mRNAs for negatively acting hnRNP proteins is triggered by splicing repression, a polarity consistent with widespread homeostatic control of splicing regulator gene expression.
    [Show full text]
  • A Systems Biology View of the Spliceosome Component Phf5a in Relation to Estrogen and Cancer
    ien r Sc ce & te S u y p s t m e o m C s Journal of f B o Vallabhu et al., J Comput Sci Syst Biol 2014, 7:6 i l o a l o n r g u y DOI: 10.4172/jcsb.1000156 o J Computer Science & Systems Biology ISSN: 0974-7230 Research Article Article OpenOpen Access Access A Systems Biology View of the Spliceosome Component Phf5a in Relation to Estrogen and Cancer Rishu Vallabhu, Eva Falck and Angelica Lindlöf* Systems Biology Research Centre, University of Skövde, Box 408, 541 28 Skövde, Sweden Abstract Cancer is a broad term for a wide spectrum of diseases and which involves the alteration in expression levels of several hundreds of genes. As such, the study of the disease from a systems biology point of view becomes rational, as the properties of a system as a whole may be very different from the properties of its individual components. However, understanding a network at the systems level not only requires knowledge about the components of the network, but also the interactions between them. Here, a systems biology view of the rat PHD finger protein 5A (Phf5a) gene was attempted; a gene previously identified as aberrantly expressed in estrogen dependent endometrial adenocarcinoma tumors from both rat and human. Phf5ais a highly conserved cysteine rich (C4HC3) zinc finger and such proteins predominantly have a role in chromatin mediated transcriptional regulation. Moreover, PHF5A is a component of the macromolecular complex spliceosome that takes part in pre-mRNA splicing and spliceosome component coding genes have previously been shown to be implicated in various cancer types and suggested to potentially be novel antitumor drugs.
    [Show full text]