DOCSLIB.ORG

Measurement of Ligand-Receptor Interactions (Biomembrane Interactions/Adhesion/Biotin/Streptavidin) CHRISTIANE A

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Measurement of Ligand-Receptor Interactions (Biomembrane Interactions/Adhesion/Biotin/Streptavidin) CHRISTIANE A Proc. Natl. Acad. Sci. USA Vol. 88, pp. 8169-8173, September 1991 Biochemistry Measurement of ligand-receptor interactions (biomembrane interactions/adhesion/biotin/streptavidin) CHRISTIANE A. HELM*t, WOLFGANG KNOLL*§, AND JACOB N. ISRAELACHVILI* *Department of Chemical and Nuclear Engineering, and Materials Department, University of California, Santa Barbara, CA 93106; and tMax-Planck-Institut for Polymerforschung, Postfach 3148, D-6500 Mainz, Federal Republic of Germany Communicated by Charles P. Bean, June 10, 1991 ABSTRACT One distinguishing feature of "life" is that deposition of a layer of L-a-dilauroylphosphatidylethanol- the physical forces between biological molecules and membrane amine (DLPE; Sigma) of area 0.55 nm2 per molecule (con- surfaces are often highly specific, in contrast to nonspecific taining 5% DPPE-biotin) (Molecular Probes), thereby expos- interactions such as van der Waals, hydrophobic, and electro- ing one biotin ligand group per 11 nm2. This deposition was static (Coulombic) forces. We have used the surface-forces- done at 30TC from a 1 mM NaCl solution at a pressure ofabout apparatus technique to study the specific "lock and key" or 38 mN/m. "ligand-receptor" interaction between two model biomem- An avidin surface was prepared from a biotin surface by brane surfaces in aqueous solution. The membranes were lipid adsorbing soluble streptavidin molecules (kindly provided by bilayers supported on mica surfaces; one carrying streptavidin Boehringer Mannheim) from an aqueous solution, thereby receptors, the other exposing biotin ligand groups. We found yielding a surface with the same density ofunsaturated avidin that, although no unusual or specific interaction occurs be- (receptor) sites as biotin (ligand) groups. Avidin surfaces tween two avidin or two biotin surfaces, an avidin and a biotin could be prepared either before they were installed in the surface exhibit a very strong, very short-range (<1 nm) surface forces apparatus or after installation inside the cham- attraction and that the binding mechanism involves equally ber. specific molecular rearrangements. The results also show that After the biotin and/or avidin surfaces were prepared, they highly specific biological interactions such as are involved in were transferred (under water) into the surface forces appa- immunological recognition and cell-cell contacts may be stud- ratus chamber previously filled with a 1 mM NaCl solution ied at the molecular level and in real time by the surface-forces- saturated with DLPE at the critical micelle concentration. apparatus technique. This was to ensure that no DLPE desorbed during the course of an experiment (10-12). To ensure also that the second During the past 20 years the fundamental forces between monolayer remained in the fluid state, all measurements were surfaces in liquids have been systematically studied both done at 33 + 1TC, which is well above the phase transition experimentally and theoretically (1). These forces-e.g., van temperature of DLPE bilayers (30.50C). The force laws, der Waals, electrostatic, hydrophobic-are not system- adhesion energies, and surface deformations were measured specific in the sense that the interaction potential is a known as previously described (8-13) between the following sur- function of distance, such as a power law or an exponential faces shown in Fig. 2: biotin-biotin, avidin-avidin, and function. Less studied is a specific binding mechanism be- avidin-biotin. tween certain molecules that have a perfect geometrical fit. Such "lock and key" or "ligand-receptor" interactions (2, 3) give rise to very strong physical-as opposed to chemical, RESULTS AND DISCUSSION covalent or metal chelating-bonds with minimal expendi- The force law between two biotin surfaces in 1 mM NaCl ture of energy. Such noncovalent bonds are central to a solution at pH -5.8 is shown in Fig. 3 Inset. The force was molecular understanding of biological recognition and mo- purely repulsive, and no adhesion was measured at any lecular engineering applications (4-7). We have studied the separation, including contact. The measured repulsion of interactions between biotin ligands and streptavidin recep- exponential decay length of -9 nm is that characteristic ofan tors (5, 7). This is one of the most-thoroughly studied electrostatic "double-layer" interaction between two sur- ligand-receptor systems, with a binding constant of 88 kJ/ faces in 1 mM monovalent electrolyte solution (1), where mol (-35 kT per bond) that is one of the highest known. each surface has a surface potential of 37 + 4 mV, corre- sponding to a surface charge density of 1 unit charge per 55 MATERIALS AND METHODS nm2. Since pure DLPE is uncharged and exhibits no double- layer repulsion (10-12), the negative surface charge must A surface-forces apparatus (8, 9) was used to measure reside on the anionic directly the forces between mica surfaces coated with lipid phosphate groups (pK 3) of the bilayers whose outer fluid monolayers contained either the biotinylated lipid molecules (see Fig. 1). Assuming that none ligand L-a-dipalmitoylphosphatidylethanolamine-biotin of the charged DPPE-biotin had gone into solution, the (DPPE-biotin) or streptavidin (the protein receptor) as measured surface charge density indicates that 20% (or more) shown in Fig. 1. For convenience and simplicity we shall call ofthe biotin groups are dissociated-a value that is typical for these surfaces the "biotin" surface and the "avidin" surface. surfactant head groups in electrolyte solutions (14). We The Langmuir-Blodgett deposition technique (10-12) was therefore conclude that the interaction between two biotin used to prepare these surfaces. First, a primary close-packed surfaces exhibits no unusual or "specific" features. monolayer of DPPE (Sigma) of area 0.42 nm2 per molecule was deposited on a mica surface. This was followed by the Abbreviations: DPPE, L-a-dipalmitoylphosphatidylethanolamine; DLPE, L-a-dilauroylphosphatidylethanolamine. tPresent address: Institut fur Physikalische Chemie, Johannes The publication costs of this article were defrayed in part by page charge Gutenberg Universitit, Jakob-Welder-Weg 11, D-6500 Mainz, Fed- payment. This article must therefore be hereby marked "advertisement" eral Republic of Germany. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 8169 Downloaded by guest on October 1, 2021 8170 Biochemistry: Helm et al. Proc. Natl. Acad. Sci. USA 88 (1991) Biotin surface r v. Mica substrate 2.7 nm n m SolutionSoltin ... ._..--------------------------- ----.-- -.- - - --. Streptavidin- 4.5 n FIG. 1. Schematic illustration .~~~~~~~~~~~~~~~~~~~~~~~~~~~. ofthe two types ofsurfaces used in r-- NH the experiments. The biotin sur- face exposes a fluid DLPE mono- layer containing DPPE-biotin lig- DLPE and molecules (5, 7) at a surface DPPE-- F w Ho A6d 9uL % a %-# W coverage of -5%. The avidin sur- biotin KNI face exposes the 4.5-nm-thick molecule 1 streptavidin receptor proteins, Ii-P-iliA each of which has four binding l11 sites for biotin-two on either side I _ _ F' of the molecule (6). The thick- .. ... nesses shown are estimates only; K . t1 I L _- __ in particular, the 4.5-nm-thick Avidin surface streptavidin molecule can easily be compressed to 3.5 nm (see Fig. 3). The force law between two avidin surfaces is shown in Fig. required a wait of about 10-20 min between force runs to 3. These surfaces were prepared..............by first .............................installing two . .... reach equilibrium (dashed line in Fig. 3). Since these maxima identical biotin surfaces in the apparatusF.... ........... ............and then, .. ..after ... ....the . represent the force needed to separate and intercalate the forces between them were measured, injecting a small quan- avidin-biotin complexes over an area of roughly irr2 10-11 tity (100 1l) of 0.6 ,uM streptavidin_solution between them. m2 (as measured optically¶), we estimate that the lateral Assuming the same adsorption kinetics as previously mea- diffusion constant for the biotin-avidin complex within the sured for a similar system (15), this procedure should have outer supported monolayer is Ddff = r2/t 10-10 cm2/s, produced an equilibrated streptavidin-saturated avidin sur- which is close to typical values previously measured for face after about 30 min. Identical force curves were measured proteins in membranes (17, 18). 2 and 5 hr after injection. If the measured adhesion force of FIR = -0.4 mN/m When the two avidin surfaces were brought together (Fig. between two contacting surfaces of close-packed avidin 3), they first experienced a weakly attractive van der Waals molecules separated by an aqueous film of thickness AD = force, resulting in an instability that caused them to "jump 1.2 nm (Fig. 3 and the left schematic of Fig. 2B) is due to van in" from D = 14 nm to the outermost weakly adhesive force der Waals forces, the Hamaker constant A, given by the minimum at D = 9 nm (cf. the left schematic in Fig. 2B). On equation lFIR = -A/6AD2, would be A 4 x 10-21 J. This further approach the two surfaces then experienced a repul- can be compared with previously measured values of A sion reflecting the unfavorable work needed to compress the (1.5-7.0) x 10-21 J for uncharged bilayers (10, 11, 21, 22) and system (aqueous gap, compressible protein, and fluid mono- A 10-20 J for adsorbed proteins (23) interacting across layers). The activation barrier needed to laterally part the aqueous solutions. We conclude that-as in the case of two proteins was finally overcome at D = 7 nm when a second instability occurred and another jump in to a more-adhesive 1From the shapes of the optical interference fringes used to "ob- minimum was found (cf. the right schematic in Fig. 2B). serve" the interacting surfaces, one can directly measure not only When the surfaces were separated from either force mini- the distance between the two surfaces (to +1A) but also the mum, they "jumped out".
Load more

Recommended publications
  • Functional Analysis of the Homeobox Gene Tur-2 During Mouse Embryogenesis
    Functional Analysis of The Homeobox Gene Tur-2 During Mouse Embryogenesis Shao Jun Tang A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy Graduate Department of Molecular and Medical Genetics University of Toronto March, 1998 Copyright by Shao Jun Tang (1998) National Library Bibriothèque nationale du Canada Acquisitions and Acquisitions et Bibiiographic Services seMces bibliographiques 395 Wellington Street 395, rue Weifington OtbawaON K1AW OttawaON KYAON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence alIowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distri%uteor sell reproduire, prêter' distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Functional Analysis of The Homeobox Gene TLr-2 During Mouse Embryogenesis Doctor of Philosophy (1998) Shao Jun Tang Graduate Department of Moiecular and Medicd Genetics University of Toronto Abstract This thesis describes the clonhg of the TLx-2 homeobox gene, the determination of its developmental expression, the characterization of its fiuiction in mouse mesodem and penpheral nervous system (PNS) developrnent, the regulation of nx-2 expression in the early mouse embryo by BMP signalling, and the modulation of the function of nX-2 protein by the 14-3-3 signalling protein during neural development.
    [Show full text]
  • The Transcriptional Activator PAX3–FKHR
    Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press The transcriptional activator PAX3–FKHR rescues the defects of Pax3 mutant mice but induces a myogenic gain-of-function phenotype with ligand-independent activation of Met signaling in vivo Frédéric Relaix,1 Mariarosa Polimeni,2 Didier Rocancourt,1 Carola Ponzetto,3 Beat W. Schäfer,4 and Margaret Buckingham1,5 1CNRS URA 2375, Department of Developmental Biology, Pasteur Institute, 75724 Paris Cedex 15, France; 2Department of Experimental Medicine, Section of Anatomy, University of Pavia, 27100 Pavia, Italy; 3Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, 10126 Turin, Italy; 4Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zurich, CH-8032 Zurich, Switzerland Pax3 is a key transcription factor implicated in development and human disease. To dissect the role of Pax3 in myogenesis and establish whether it is a repressor or activator, we generated loss- and gain-of-function alleles by targeting an nLacZ reporter and a sequence encoding the oncogenic fusion protein PAX3–FKHR into the Pax3 locus. Rescue of the Pax3 mutant phenotypes by PAX3–FKHR suggests that Pax3 acts as a transcriptional activator during embryogenesis. This is confirmed by a Pax reporter mouse. However, mice expressing PAX3–FKHR display developmental defects, including ectopic delamination and inappropriate migration of muscle precursor cells. These events result from overexpression of c-met, leading to constitutive activation of Met signaling, despite the absence of the ligand SF/HGF. Haploinsufficiency of c-met rescues this phenotype, confirming the direct genetic link with Pax3. The gain-of-function phenotype is also characterized by overactivation of MyoD.
    [Show full text]
  • Genomic Targeting of Epigenetic Probes Using a Chemically Tailored Cas9 System
    Genomic targeting of epigenetic probes using a chemically tailored Cas9 system Glen P. Liszczaka, Zachary Z. Browna, Samuel H. Kima, Rob C. Oslunda, Yael Davida, and Tom W. Muira,1 aDepartment of Chemistry, Princeton University, Princeton, NJ 08544 Edited by James A. Wells, University of California, San Francisco, CA, and approved December 13, 2016 (received for review September 20, 2016) Recent advances in the field of programmable DNA-binding proteins Here, we report a method that combines the versatility of have led to the development of facile methods for genomic pharmacologic manipulation with the specificity of a genetically localization of genetically encodable entities. Despite the extensive programmable DNA-binding protein. Our strategy uses a utility of these tools, locus-specific delivery of synthetic molecules chemically tailored dCas9 to display a pharmacologic agent at a remains limited by a lack of adequate technologies. Here we combine genetic locus of interest in live mammalian cells (Fig. 1). This is trans the flexibility of chemical synthesis with the specificity of a pro- accomplished using split intein-mediated protein -splicing grammable DNA-binding protein by using protein trans-splicing to (PTS) to site-specifically link a recombinant dCas9:guide RNA ligate synthetic elements to a nuclease-deficient Cas9 (dCas9) (gRNA) complex to the synthetic cargo of choice (22). Indeed, in vitro and subsequently deliver the dCas9 cargo to live cells. we show that the remarkable specificity, efficiency, and speed of PTS allow the direct generation of desired dCas9 conjugates The versatility of this technology is demonstrated by delivering within the cell culture media, thereby facilitating a streamlined dCas9 fusions that include either the small-molecule bromodomain “one-pot” approach for genomic targeting of the reaction product.
    [Show full text]
  • Geometry, Epistasis, and Developmental Patterning
    Geometry, epistasis, and developmental patterning Francis Corson and Eric Dean Siggia1 Center for Studies in Physics and Biology, The Rockefeller University, New York, NY 10021 This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected in 2009. Contributed by Eric Dean Siggia, February 6, 2012 (sent for review November 28, 2011) Developmental signaling networks are composed of dozens of (5) shows that even differentiation can be reversed. Yet they have components whose interactions are very difficult to quantify in provided a useful guide to experiments. an embryo. Geometric reasoning enumerates a discrete hierarchy These concepts admit a natural geometric representation, of phenotypic models with a few composite variables whose para- which can be formalized in the language of dynamical systems, meters may be defined by in vivo data. Vulval development in also called the geometric theory of differential equations (Fig. 1). ’ the nematode Caenorhabditis elegans is a classic model for the in- When the molecular details are not accessible, a system s effec- tegration of two signaling pathways; induction by EGF and lateral tive behavior may be represented in terms of a small number of signaling through Notch. Existing data for the relative probabilities aggregate variables, and qualitatively different behaviors enum- of the three possible terminal cell types in diverse genetic back- erated according to the geometrical structure of trajectories or grounds as well as timed ablation of the inductive signal favor topology. The fates that are accessible to a cell are associated with attractors—the valleys in Waddington’s “epigenetic landscape” one geometric model and suffice to fit most of its parameters.
    [Show full text]
  • Genetic Dissection of Developmental Pathways*§ †
    Genetic dissection of developmental pathways*§ † Linda S. Huang , Department of Biology, University of Massachusetts-Boston, Boston, MA 02125 USA Paul W. Sternberg, Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena, CA 91125 USA Table of Contents 1. Introduction ............................................................................................................................1 2. Epistasis analysis ..................................................................................................................... 2 3. Epistasis analysis of switch regulation pathways ............................................................................ 3 3.1. Double mutant construction ............................................................................................. 3 3.2. Interpretation of epistasis ................................................................................................ 5 3.3. The importance of using null alleles .................................................................................. 6 3.4. Use of dominant mutations .............................................................................................. 7 3.5. Complex pathways ........................................................................................................ 7 3.6. Genetic redundancy ....................................................................................................... 9 3.7. Limits of epistasis ......................................................................................................
    [Show full text]
  • Cytokine Engineering for Improved Ligand/Receptor Traff Ming Dynamics Douglas a Lauffenburger, Eric M Fallon and Jason M Haugh
    Review R257 Scratching the (cell) surface: cytokine engineering for improved ligand/receptor traff Ming dynamics Douglas A Lauffenburger, Eric M Fallon and Jason M Haugh Cytokines can be engineered for greater potency in stimulating Cytokine engineering cellular functions. An obvious test criterion for an improved Recombinant DNA methods offer the possibility of pro- cytokine is receptor-binding affinity, but this does not always ducing cytokines possessing properties superior to those correlate with improved biological response. By combining of the natural. wild-type proteins. In aitrr, as well as in U&XI protein-engineering techniques with studies of receptor applications beckon. Mammalian cell binreactors, for trafficking and signaling, it might be possible to identify the example, for production of pharmacological proteins or ligand receptor-binding properties that should be sought. expansion of differentiated cell populations, often require feeding with specific cytokines for the ximulation of cell Address: Division of Bioengineering & Environmental Health, Department of Chemical Engineering and Center for Biomedical proliferation or differentiation. or the prevention of apop- Engineering, Massachusetts Institute of Technology, Cambridge tosis [1,2]. Typically, periodic re-feeding is required, as a MA 02139, USA. result of metabolic degradation of the cytokines arising from the action of a combination of extracellular and intra- Correspondence: Douglas A Lauffenburger E-mail: [email protected] cellular proteases [3]. Creation of cytokinc
    [Show full text]
  • In Vivo and in Vitro Analysis of Dll1 and Pax6 Function in the Adult Mouse Pancreas
    TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Experimentelle Genetik In vivo and in vitro analysis of Dll1 and Pax6 function in the adult mouse pancreas Davide Cavanna Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. D. Langosch Prüfer der Dissertation: 1. Univ.-Prof. Dr. M. Hrabé de Angelis 2. Univ.-Prof. A. Schnieke, Ph.D. Die Dissertation wurde am 03.07.2013 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 10.12.2013 angenommen. I. Table of contents I. TABLE OF CONTENTS .................................................................................................. I II. FIGURES AND TABLES ................................................................................................ V III. ABBREVIATIONS ................................................................................................. VIII IV. PUBLICATIONS, TALKS, AND POSTERS ................................................................... XI V. ACKNOWLEDGMENTS .............................................................................................. XII VI. AFFIRMATION ..................................................................................................... XIV 1. SUMMARY/ZUSAMMENFASSUNG ............................................................................
    [Show full text]
  • The Many Faces of Pleiotropy
    Opinion The many faces of pleiotropy Annalise B. Paaby and Matthew V. Rockman Department of Biology and Center for Genomics & Systems Biology, New York University, 12 Waverly Place, New York, NY 10003, USA Pleiotropy is the well-established phenomenon of a pleiotropy is a feature of the genotype–phenotype map single gene affecting multiple traits. It has long played that defines the genetic and evolutionary autonomy of a central role in theoretical, experimental, and clinical aspects of phenotype, independent of fitness. This is the research in genetics, development, molecular biology, mutational pleiotropy underlying the diverse manifesta- evolution, and medicine. In recent years, genomic tech- tions of syndromic diseases, the ontogenetic pleiotropy that niques have brought data to bear on fundamental ques- underlies classical questions about allometry and hetero- tions about the nature and extent of pleiotropy. chrony, and the molecular pleiotropy that underlies ques- However, these efforts are plagued by conceptual diffi- tions about relative importance of cis-regulatory versus culties derived from disparate meanings and interpreta- protein-coding variants. tions of pleiotropy. Here, we describe distinct uses of the In ‘selectional pleiotropy’, the question is about the pleiotropy concept and explain the pitfalls associated number of separate components of fitness a mutation with applying empirical data to them. We conclude that, affects. In some cases, the multiple fitness components for any question about the nature or extent of pleiotropy, are life-history traits of a single individual, which is at the appropriate answer is always ‘What do you mean?’. the heart of the antagonistic pleiotropy model for the evolution of aging [1].
    [Show full text]
  • The EGF Receptor Ligand Amphiregulin Controls Cell Division Via Foxm1
    Oncogene (2016) 35, 2075–2086 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc ORIGINAL ARTICLE The EGF receptor ligand amphiregulin controls cell division via FoxM1 SW Stoll1, PE Stuart1, WR Swindell1, LC Tsoi2,BLi3, A Gandarillas4, S Lambert1, A Johnston1, RP Nair1 and JT Elder1,5 Epidermal growth factor receptor (EGFR) is central to epithelial cell physiology, and deregulated EGFR signaling has an important role in a variety of human carcinomas. Here we show that silencing of the EGF-related factor amphiregulin (AREG) markedly inhibits the expansion of human keratinocytes through mitotic failure and accumulation of cells with ⩾ 4n DNA content. RNA-sequencing- based transcriptome analysis revealed that tetracycline-mediated AREG silencing significantly altered the expression of 2331 genes, 623 of which were not normalized by treatment with EGF. Interestingly, genes irreversibly upregulated by suppression of AREG overlapped with genes involved in keratinocyte differentiation. Moreover, a significant proportion of the irreversibly downregulated genes featured upstream binding sites recognized by forkhead box protein M1 (FoxM1), a key transcription factor in the control of mitosis that is widely dysregulated in cancer. The downregulation of FoxM1 and its target genes preceded mitotic arrest. Constitutive expression of FoxM1 in AREG knockdown cells normalized cell proliferation, reduced the number of cells with ⩾ 4n DNA content and rescued expression of FoxM1 target genes. These results demonstrate that AREG controls G2/M progression and cytokinesis in keratinocytes via activation of a FoxM1-dependent transcriptional program, suggesting new avenues for treatment of epithelial cancer. Oncogene (2016) 35, 2075–2086; doi:10.1038/onc.2015.269; published online 3 August 2015 INTRODUCTION a variety of epithelial neoplasms and it may confer tumor cells 31–34 Epidermal homeostasis requires balance between keratinocyte with autonomous growth.
    [Show full text]
  • Hedgehog Signaling: Networking to Nurture a Promalignant Tumor Microenvironment
    Published OnlineFirst July 20, 2011; DOI: 10.1158/1541-7786.MCR-11-0175 Molecular Cancer Subject Review Research Hedgehog Signaling: Networking to Nurture a Promalignant Tumor Microenvironment Lillianne G. Harris, Rajeev S. Samant, and Lalita A. Shevde Abstract In addition to its role in embryonic development, the Hedgehog pathway has been shown to be an active participant in cancer development, progression, and metastasis. Although this pathway is activated by autocrine signaling by Hedgehog ligands, it can also initiate paracrine signaling with cells in the microenvironment. This creates a network of Hedgehog signaling that determines the malignant behavior of the tumor cells. As a result of paracrine signal transmission, the effects of Hedgehog signaling most profoundly influence the stromal cells that constitute the tumor microenvironment. The stromal cells in turn produce factors that nurture the tumor. Thus, such a resonating cross-talk can amplify Hedgehog signaling, resulting in molecular chatter that overall promotes tumor progression. Inhibitors of Hedgehog signaling have been the subject of intense research. Several of these inhibitors are currently being evaluated in clinical trials. Here, we review the role of the Hedgehog pathway in the signature characteristics of cancer cells that determine tumor development, progression, and metastasis. This review condenses the latest findings on the signaling pathways that are activated and/or regulated by molecules generated from Hedgehog signaling in cancer and cites promising clinical interventions. Finally, we discuss future directions for identifying the appropriate patients for therapy, developing reliable markers of efficacy of treatment, and combating resistance to Hedgehog pathway inhibitors. Mol Cancer Res; 9(9); 1165–74.
    [Show full text]
  • Inducible Short-Term and Stable Long-Term Cell Culture Systems Reveal That the PAX3-FKHR Fusion Oncoprotein Regulates CXCR4, PAX3, and PAX7 Expression
    Laboratory Investigation (2004) 84, 1060–1070 & 2004 USCAP, Inc All rights reserved 0023-6837/04 $30.00 www.laboratoryinvestigation.org Inducible short-term and stable long-term cell culture systems reveal that the PAX3-FKHR fusion oncoprotein regulates CXCR4, PAX3, and PAX7 expression Oana Tomescu1, Shujuan J Xia1, Donna Strezlecki1, Jeannette L Bennicelli1,*, Jill Ginsberg1,w, Bruce Pawel2 and Frederic G Barr1 1Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA and 2Department of Pathology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA In the pediatric cancer alveolar rhabdomyosarcoma (ARMS), the 2;13 chromosomal translocation juxtaposes the PAX3 and FKHR genes to generate a chimeric transcription factor. To explore molecular pathways altered by this oncoprotein, we generated an inducible form by fusing PAX3-FKHR to a modified estrogen receptor ligand-binding domain and expressed this construct in the RD embryonal rhabdomyosarcoma cell line. This inducible system permits short-term evaluation of downstream expression targets of PAX3-FKHR and complements a panel of stable long-term RD subclones constitutively expressing PAX3-FKHR. Using these two sets of resources, we investigated several candidate PAX3-FKHR target genes. First, we demonstrated in both short-term and long-term systems that PAX3-FKHR upregulates expression of the gene encoding the chemokine receptor CXCR4. In addition, we found that expression of wild-type PAX3 is upregulated, whereas expression of wild-type PAX7 is downregulated by PAX3-FKHR. In the presence of cycloheximide, CXCR4 and PAX3 are still inducible, supporting the hypothesis that these genes are direct transcriptional targets of PAX3- FKHR.
    [Show full text]
  • The Development and Evolutionary Origin of Barbels in the Channel
    THE DEVELOPMENT AND EVOLUTIONARY ORIGIN OF BARBELS IN THE CHANNEL CATFISH ICTALURUS PUNCTATUS (SILURIFORMES: ICTALURIDAE) by MICHAEL BRENT HAWKINS B.A., University of Colorado, 2008 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirement for the degree of Master’s of Arts Department of Ecology and Evolutionary Biology 2011 This thesis entitled: The development and evolutionary origin of barbels in the channel catfish Ictalurus punctatus (Siluriformes: Ictaluridae) written by Michael Brent Hawkins has been approved for the Department of Ecology and Evolutionary Biology David W. Stock, committee co-chair Alexander Cruz, committee co-chair Date The final copy of this thesis has been examined by the signatories, and we Find that both the content and the form meet acceptable presentation standards Of scholarly work in the above mentioned discipline. Hawkins, Michael Brent (M.A., Ecology and Evolutionary Biology) The development and evolutionary origin of barbels in the channel catfish Ictalurus punctatus (Siluriformes: Ictaluridae) Thesis co-directed by Professor Alexander Cruz and Associate Professor David Stock Understanding the origin of morphological novelties is an important goal of evolutionary developmental biology. In pursuit of this goal, we have examined the developmental genetic mechanisms that underlie growth and patterning in a largely overlooked group of morphological novelties: the barbels of fishes. Barbels are appendages that project from the head region in a large and disparate assortment of fish taxa, ranging from hagfishes to gobies. They often bear sensory organs and can be supported by a rod of connective tissue, muscle, cartilage, or bone.
    [Show full text]