DOCSLIB.ORG

(DHEA) on Human Neural Stem Cell Cultures Derived from the Fetal Cortex

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
(DHEA) on Human Neural Stem Cell Cultures Derived from the Fetal Cortex Mitotic and neurogenic effects of dehydroepiandrosterone (DHEA) on human neural stem cell cultures derived from the fetal cortex Masatoshi Suzuki*†, Lynda S. Wright*, Padma Marwah‡, Henry A. Lardy‡, and Clive N. Svendsen*§ *Departments of Anatomy and Neurology and the Waisman Center, University of Wisconsin, 1500 Highland Avenue, Madison, WI 53705-2280; †Department of Veterinary Physiology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; and ‡Department of Biochemistry and Institute for Enzyme Research, University of Wisconsin, 1710 University Avenue, Madison, WI 53726 Contributed by Henry A. Lardy, December 29, 2003 Dehydroepiandrosterone (DHEA) is a neurosteroid with potential very low (6, 11). Thus, there is some question as to the relevance effects on neurogenesis and neuronal survival in humans. How- of the effect of DHEA on rodents and how this relates to the ever, most studies on DHEA have been performed in rodents, and human situation. there is little direct evidence for biological effects on the human Recent studies have shown that neural precursor cells can be nervous system. Furthermore, the mechanism of its action is derived from the developing brain allowing the direct examina- unknown. Here, we show that DHEA significantly increased the tion of proliferation, differentiation, and migration of these cells growth rates of human neural stem cells derived from the fetal in the culture dish (12, 13). These cells can be maintained in cortex and grown with both epidermal growth factor (EGF) and culture as spherical aggregates termed neurospheres (14–16) leukemia inhibitory factor (LIF). However, it had no effect on that consist of both multipotent stem cells and more restricted cultures grown in either factor alone, suggesting a specific action progenitor cells responsive to the mitogens epidermal growth on the EGF͞LIF-responsive cell. Precursors of DHEA such as preg- factor (EGF) and fibroblast growth factor 2 (FGF-2) (17). nenolone or six of its major metabolites, had no significant effect Neurospheres are regionally specified based on the brain region on proliferation rates. DHEA did not alter the small number (<3%) from which they were isolated (18, 19) and also differ between of newly formed neuroblasts or the large number (>95%) of species (20). However, recently, we have grown neurospheres nestin-positive precursors. However, the number of glial fibrillary from the human fetal cortex for extended periods of time in EGF acidic protein-positive cells, its mRNA, and protein were signifi- and LIF and shown that they represent a fairly homogenous cantly increased by DHEA. We found both N-methyl-D-aspartate population of cells immunoreactive for the stem cell marker and sigma 1 antagonists, but not GABA antagonists, could com- nestin (21, 22). After a full genomic analysis of these cells, we pletely eliminate the effects of DHEA on stem cell proliferation. have shown them to be stable in culture over time and between Finally we asked whether the EGF͞LIF͞DHEA-responsive stem cells lines with regard to global gene expression and termed them had an increased potential for neurogenesis and found a 29% long-term human neural stem cells derived from the cortex increase in neuronal production when compared to cultures grown (ltNSCCTX) (22). in EGF͞LIF alone. Together these data suggest that DHEA is in- Given the species differences in DHEA and stem cell biology, volved in the maintenance and division of human neural stem cells. ltNSCCTX provide an ideal and innovative model to understand Given the wide availability of this neurosteroid, this finding has developmental and molecular mechanisms of neurosteroid ac- important implications for future use. tions in the human central nervous system. We show here that DHEA, but not its derivatives, increased the cell proliferation of ehydroepiandrosterone (DHEA) and its sulfate are the ltNSCCTX in vitro. We also established which receptor signaling Dmost abundant steroids in the blood of young adult humans. mediated this DHEA action, and finally showed that ltNSCCTX Levels of DHEA peak at Ϸ20 years of age and then decline to grown in the presence of DHEA underwent increased neuro- reach values of 20–30% at 70–80 years of age (1). Significant genesis. These results have important implications for brain interest has arisen from the hypothesis that declining DHEA repair and are relevant to understanding the actions of DHEA concentrations in adults may serve as an indicator of a number on the human brain. of conditions including the loss of insulin sensitivity, obesity, diabetes, cardiovascular diseases, stress, and aging (2). In clinical Materials and Methods studies, DHEA levels were found to decline in mental illnesses Neuronal Progenitor Cell Culture. Human embryonic tissue was such as major depressive disorder or in systematic diseases that obtained from the Birth Defects Laboratory at the University of respond to DHEA supplementation (3, 4). Washington, Seattle. Neurospheres were generated from three While DHEA represents the most abundant steroid product of cortex samples, M006 (13 weeks postconception male), M007 (18 the adrenal cortex, it has also been identified as a ‘‘neuroste- weeks postconception male), and M009 (16 weeks postconcep- roid.’’ Neurosteroids are synthesized de novo in the central tion male). The method of collection conformed to the guide- nervous system independent, at least in part, of peripheral organ lines recommended by National Institutes of Health for the activity (5, 6). Neurosteroids possess the ability to affect neurons collection of such tissues and set out by the University of ␥ through activation of -aminobutyric acid (GABA)A, N-methyl- Washington and the University of Wisconsin, Madison. Institu- D-aspartate (NMDA), and sigma receptors (5, 6), although the exact role of each with regard to DHEA is not well established. DHEA has been shown to be neuroprotective after oxidative Abbreviations: DHEA, dehydroepiandrosterone; EGF, epidermal growth factor; GABA, ␥-aminobutyric acid; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; stress in rat hippocampal neuronal cultures (7) and hippocampal ltNSCCTX, long-term cortical neural stem cells; MK801, (ϩ)-5-methyl-10,11-dihydro-5H- damage induced by NMDA (8). DHEA has also been shown to dibenzo[a,d]cyclohepten-5,10-imine hydrogen malate; NMDA, N-methyl-D-aspartate; be a potential signaling molecule in neuronal differentiation TuJ1, ␤-tubulin III. during development (9) and has recently been shown to increase §To whom correspondence should be addressed at: T611 Waisman Center, 1500 Highland neurogenesis in the adult rodent hippocampus (10). In contrast Avenue, Madison, WI 53705. E-mail: [email protected]. to the human brain, levels of DHEA in the adult rat brain are © 2004 by The National Academy of Sciences of the USA 3202–3207 ͉ PNAS ͉ March 2, 2004 ͉ vol. 101 ͉ no. 9 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0307325101 Downloaded by guest on September 28, 2021 Fig. 1. Schematic describing culture schedules and treatments for ltNSCctx. Neurospheres were generated from three fetal cortex samples, initially grown for 4 weeks in EGF and FGF-2. The cultures were then switched to medium supplemented with EGF alone. After Ͼ10 weeks of growth, the cultures were supplemented with EGF and LIF and maintained for a further 4 weeks. Thirty to 40 neurospheres were cultured in coated T25 flasks with basal medium in the presence or absence of EGF, LIF, and DHEA. After 9 days, neurospheres were pulsed with 0.2 ␮M BrdUrd for 14 h and then dissociated into a single cell suspension with enzyme. Dissociated cells were plated onto poly(L)-lysine͞laminin-coated coverslips for 1 h. To check the number of cells immunostained with BrdUrd or other cell markers, cells were fixed 1 h after plating. For differentiation studies, the plated cells were differentiated for 7 days under serum-free conditions and then visualized with anti-TuJ1, GFAP, and Hoechst-labeled nuclei. tional Review Board approval was obtained for all of these Systems) were used at a final concentration of 10 ng͞ml. The studies. NMDA receptor antagonist (ϩ)-5-methyl-10,11-dihydro-5H- The schematic illustration of the experimental schedule and dibenzo[a,d]cyclohepten-5,10-imine hydrogen malate (MK801) ␮ treatments is summarized in Fig. 1. Cortical progenitors were and GABAA receptor antagonist bicuculline (10 M each in isolated from embryonic brain and induced to proliferate as medium) were purchased fromR&DSystems. Sigma 1 receptor free-floating neurospheres (16). Freshly isolated tissue was antagonists BD1063 or haloperidol (Tocris Cookson, Ellisville, mechanically chopped into small cubes using a McLwain tissue MO) were used at a final concentration of 3 ␮M. chopper (Mickle Laboratory Engineering, Surrey, England) and seeded into T75 flasks at an approximate density of 200,000 cells Immunocytochemistry. Cultures were fixed in ice cold methanol (eight cubes) per ml of basal medium [DMEM͞Ham’s F-12 (7:3) for 20 min or 4% paraformaldehyde (PFA in PBS) at room containing penicillin͞streptomycin͞amphotericin B (PSA, 1%)] temperature and washed in PBS. The cells were then incubated supplemented with B27 (2%; Life Technologies), EGF (20 in 2 M HCl for 20 min at 37°C. The acid was neutralized with 0.1 ng͞ml; Sigma), and FGF-2 (20 ng͞ml;R&DSystems) with M sodium borate and washed several times in PBS. Fixed cells heparin (5 ␮g͞ml). All cultures were maintained in a humidified were blocked in 5% goat serum with 0.3% Triton X-100 and incubator (37°C) and half the growth medium was replenished incubated with anti-BrdUrd antibodies (rat monoclonal, 1:500, every 4–5 days. For passaging of neurospheres, a chopping Accurate Chemical, Westbury, NY) for 30 min. After incubation method was used that does not require trypsin or mechanical with the primary antibodies, either fluorescein or Cy3 (goat dissociation, and cell͞cell contact was continuously maintained anti-rat IgG, 1:1,000, The Jackson Laboratory) was used to (16). Neurospheres were passaged every 14 days by chopping visualize the signal.
Load more

Recommended publications
  • Growth Hormone Enhances Hepatic Epidermal Growth Factor Receptor Concentration in Mice
    Growth hormone enhances hepatic epidermal growth factor receptor concentration in mice. J O Jansson, … , W G Beamer, L A Frohman J Clin Invest. 1988;82(6):1871-1876. https://doi.org/10.1172/JCI113804. Research Article The effect of growth hormone (GH) on binding of epidermal growth factor (EGF) to liver membrane preparations was investigated in hypophysectomized mice and partially GH-deficient, genetic mutant "little" (lit/lit) mice. The EGF binding of normal male mice and testosterone-treated females was higher than in normal females. Due to diminished receptor concentration, hepatic EGF binding was decreased in male and female lit/lit mice to a level that was unaffected by gender or androgen treatment. GH replacement therapy by intermittent injections and continuous infusion restored the EGF binding of hypophysectomized mice to normal male and female levels, respectively, suggesting a role for the more pulsatile GH secretion in normal males. In lit/lit mice, however, both continuous and intermittent GH resulted in EGF binding levels comparable to those in normal females. In normal males continuous GH suppressed EGF binding. In conclusion, endogenous GH secretion induces EGF receptors in mice and this effect may be modulated by sex differences in GH secretion. Find the latest version: https://jci.me/113804/pdf Growth Hormone Enhances Hepatic Epidermal Growth Factor Receptor Concentration in Mice John-Olov Jansson,** Staffan Ekberg,t Steven B. Hoath,* Wesley G. Beamer," and Lawrence A. Frohman* Divisions of*Endocrinology and ONeonatology, University of Cincinnati College ofMedicine, Cincinnati, Ohio 45267; "Jackson Laboratory, Bar Harbor, Maine 04609; and tDepartment ofPhysiology, University ofGoteborg, Sweden Abstract (IGF-I), which may function in a paracrine and autocrine as well as endocrine manner (6-8).
    [Show full text]
  • The Endometrial Lymphatic Vasculature: Function and Dysfunction
    The Endometrial Lymphatic Vasculature: Function and Dysfunction Jane E. Girling and Peter A.W. Rogers Gynaecology Research Centre, Department of Obstetrics and Gynaecology, The University of Melbourne, Melbourne, Victoria 3052, Australia. Corresponding Author: Dr Jane E Girling Gynaecology Research Centre Department of Obstetrics and Gynaecology The University of Melbourne The Royal Women’s Hospital Cnr Flemington Rd and Grattan St Parkville, VIC 3058, Australia Email: [email protected] Telephone: +61 3 8345 3721 Fax: +61 3 8345 3702 1 Abstract The endometrium has a complex and dynamic blood and lymphatic vasculature which undergoes regular cycles of growth and breakdown. While we now have a detailed picture of the endometrial blood vasculature, our understanding of the lymphatic vasculature in the endometrium is limited. Recent studies have illustrated that the endometrium contains a population of lymphatic vessels with restricted distribution in the functional layer relative to the basal layer. The mechanisms responsible for this restricted distribution and the consequences for endometrial function are not known. This review will summarise our current understanding of endometrial lymphatics, including the mechanisms regulating their growth and function. The potential contribution of lymphatic vessels and lymphangiogenic growth factors to various endometrial disorders will be discussed. Keywords: Blood Vessels, Decidua, Endometrium, Lymphatics, Menstruation, VEGFC, VEGFD 2 1. Introduction The endometrium has a complex and dynamic blood and lymphatic vasculature which undergoes regular cycles of growth and breakdown. These cyclic changes reflect variations in circulating sex steroids and uterine blood flow and result in cyclic patterns in tissue oxygenation, haemostasis, nutrient supply, fluid balance and leukocyte distribution. Appropriate growth and functioning of the vasculature is essential for normal endometrial function, including preparation for potential embryo implantation and subsequent pregnancy.
    [Show full text]
  • Involvement of Vascular Endothelial Growth Factor Signaling in CLR/RAMP1 and CLR/RAMP2-Mediated Pro-Angiogenic Effect of Interme
    289-294.qxd 18/6/2010 08:32 Ì ™ÂÏ›‰·289 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 26: 289-294, 2010 289 Involvement of vascular endothelial growth factor signaling in CLR/RAMP1 and CLR/RAMP2-mediated pro-angiogenic effect of intermedin on human vascular endothelial cells GIOVANNA ALBERTIN, ELISA SORATO, BARBARA OSELLADORE, ALESSANDRA MASCARIN, CINZIA TORTORELLA and DIEGO GUIDOLIN Department of Human Anatomy and Physiology, Section of Anatomy, University of Padova-Medical School, I-35121 Padova, Italy Received January 27, 2010; Accepted March 30, 2010 DOI: 10.3892/ijmm_00000464 Abstract. Intermedin (IMD) is a recently discovered peptide initiation and propagation by transactivating the VEGF closely related to adrenomedullin. Its principal physiological receptor-2 machinery. activity is its role in the regulation of the cardiovascular system, where it exerts a potent hypotensive effect. In addition, Introduction data were recently provided showing that this peptide is able to exert a clearcut pro-angiogenic effect both in vitro and In early 2004, a novel member of the calcitonin (CT)/calcitonin- in vivo. IMD acts through the non-selective interaction with gene related peptide (CGRP) family was independently receptor complexes formed by the dimerization of calcitonin- identified by two separate groups. Roh and colleagues (1) like receptor (CLR) with the receptor activity-modifying discovered the human form of this peptide in cells within the proteins RAMP1, 2 or 3. Thus, in the present study, the role of intermediate lobe of the pituitary and called it intermedin (IMD). CLR/RAMP complexes in mediating the pro-angiogenic effect At the same time, Takei et al (2) identified in mammals a 146- induced by IMD on human umbilical vein endothelial cells 150 amino acid prepro-hormone which yielded to a 47-amino (HUVECs) cultured on Matrigel was examined.
    [Show full text]
  • Erythropoietin Using ALZET Osmotic Pumps
    ALZET® Bibliography References on the Administration of Erythropoietin Using ALZET Osmotic Pumps Q8440: S. Dey, et al. Sex-specific brain erythropoietin regulation of mouse metabolism and hypothalamic inflammation. JCI Insight 2020;5(5): Agents: Erythropoietin, recombinant human Vehicle: Saline; Route: CSF/CNS (intracerebral); IV; Species: Mice; Pump: 2006; Duration: 14 days; ALZET Comments: Dose (3000 U/kg); Controls received mp w/ vehicle; animal info (Tg21 mice); recombinant human Erythropoietin aka recombinant human EPO; ALZET brain infusion kit 3 used; Brain coordinates (midline, 1.00 mm; antero- posterior, 0.34 mm; dorsoventral, 2.30 mm); dental cement used;replacement therapy (Erythropoietin); Q8045: E. K. Kim, et al. Local Subcutaneous Injection of Erythropoietin Might Improve Fat Graft Survival, Whereas Continuous Infusion Using an Osmotic Pump Device Was Harmful by Provoking an Overwhelming Foreign Body Reaction in a Nude Mouse Model. Archives of Aesthetic Plastic Surgery 2018;24(3):128-133 Agents: Erythropoietin Vehicle: Saline; Route: SC; Species: Mice; Pump: 1007D; Duration: 1 week; ALZET Comments: Dose (1,000 IU of EPO); animal info (36 weeks old, CD-1, Male, 20-25 g); EPO aka hemangiogenic and antiapoptotic factor ; dependence; Q4880: E. H. Sanchez-Mendoza, et al. Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions. Journal of Visualized Experiments 2016;107(1-9 ALZET Comments: Erythropoietin, recombinant human; CSF/CNS; Mice; 30 days; Controls received
    [Show full text]
  • Human Transforming Growth Factor Α (TGF-Α)
    787 STOMACH Gut: first published as 10.1136/gut.51.6.787 on 1 December 2002. Downloaded from Human transforming growth factor α (TGF-α) is digested to a smaller (1–43), less biologically active, form in acidic gastric juice T Marchbank, R Boulton, H Hansen, R J Playford ............................................................................................................................. Gut 2002;51:787–792 Background: Transforming growth factor α (TGF-α) is a 50 amino acid peptide with potent prolifera- tive and cytoprotective activity present in gastric mucosa and juice. Aims: To determine the forms and biological activity of natural and recombinant TGF-α following incu- bation with acid pepsin. Patients: Human gastric juice was obtained under basal conditions from patients taking acid suppres- sants and from volunteers undergoing intragastric neutralisation. See end of article for Methods: Samples were analysed using mass spectroscopy and/or high pressure liquid chromatogra- authors’ affiliations phy with radioimmunoassay. Biological activity was determined using thymidine incorporation into rat ....................... hepatocytes and an indomethacin/restraint induced gastric damage rat model. α α Correspondence to: Results: TGF- 1–50 is cleaved to TGF- 1–43 by acid pepsin and this is the predominant form in normal Professor R J Playford, gastric juice. However, intragastric neutralisation or taking acid suppressants caused the predominant Gastroenterology Section, α α 3 form to be TGF- 1–50.TGF- 1–43 had only half of the ability to maximally stimulate [ H]thymidine incorpo- Imperial College School of α ration into primary rat hepatocytes (28 177 (1130) DPM/well for 2.16 nM TGF- 1–43 v 63 184 (3536) Medicine, Hammersmith α Hospital Campus, Du Cane DPM/well for TGF- 1–50; p<0.001).
    [Show full text]
  • Metabolische Signaturen Des Insulin-Like Growth Factor 1 Anhand Von Metabolom-Untersuchungen in Plasma Und Urin
    Institut für Klinische Chemie und Laboratoriumsmedizin Direktor: Univ.-Prof. Dr. med. M. Nauck Universitätsmedizin der Universität Greifswald Metabolische Signaturen des Insulin-like growth factor 1 anhand von Metabolom-Untersuchungen in Plasma und Urin Inaugural-Dissertation zur Erlangung des akademischen Grades Doktor der Medizin (Dr. med.) der Universitätsmedizin der Universität Greifswald 2019 Vorgelegt von Henrike Knacke geb. am 07.02.1992 in Eutin Name des Dekans: Prof. Dr. Max P. Baur Erstgutachter: PD Dr. Nele Friedrich Zweitgutachter: Prof. Dr. Martin Reincke (München) Tag der Disputation: 29.11.2019 um 13:00 Uhr Ort der Disputation: Klinik für Innere Medizin A, Seminarraum 7.0.15/17 Universitätsmedizin Greifswald INHALTSVERZEICHNIS 1. EINLEITUNG ......................................................................................................................................... 3 1.1 Fragestellung ..................................................................................................................................... 3 1.2 Regulierung der IGF-I-Sekretion ........................................................................................................ 3 1.3 IGF-Binding Proteins .......................................................................................................................... 4 1.4 IGF-I Signalweg und Effekte ............................................................................................................... 4 1.5 Physiologische Funktionen von IGF-I und assoziierte Erkrankungen
    [Show full text]
  • Insulin As a Growth Factor
    003 1-3998/85/1909-0879$02.00/0 PEDIATRIC RESEARCH Vol. 19, No. 9, 1985 Copyright O 1985 International Pediatric Research Foundation, Inc Printed in U.S. A. Insulin as a Growth Factor D. J. HILL AND R. D. G. MILNER Departrnenl c!j'Pucdiutricc, Unive,:sitj. of Sl~effield,Cliildren :s Hospital, Shefic~ld,England ABSTRACT. Insulin is a potent mitogen for many cell attention than its well known, acute metabolic actions. Insulin types in vitro. During tissue culture, supraphysiological also can influence growth in vivo. The poor growth of a chilld concentrations of insulin are necessary to promote cell with diabetes (1) contrasts with the overgrowth of the hyperin- replication in connective or musculoskeletal tissues. Insulin sulinemic infant of a diabetic mother (2). The growth-promoting promotes the growth of these cells by binding, with low effect of insulin in vivo was demonstrated experimentally by affinity, to the type I insulin-like growth factor (IGF) Salter and Best in 1953 (3); these investigators restored growth receptor, not through the high affinity insulin receptor. In to hypophysectomized rats by treatment with insulin and a high other cell types, such as hepatocytes, embryonal carcinoma carbohydrate diet. Rats given insulin grew as well as those given cells, or mammary tumor cells, the type I IGF receptor is growth hormone but consumed substantially more food. Any virtually absent, and insulin stimulates the growth of these analysis of the action of insulin in promoting growth must clearly cells at physiological concentrations by binding to the high separate those effects which are due to anabolism resulting frc~m affinity insulin receptor.
    [Show full text]
  • Effect of Mouse Epidermal Growth Factor on Plasma Concentrations of LH, FSH and Testosterone in Rams B
    Effect of mouse epidermal growth factor on plasma concentrations of LH, FSH and testosterone in rams B. W. Brown, P. E. Mattner, B. A. Panaretto and G. H. Brown C.S.I.R.O., Division ofAnimal Production, P.O. Box 239, Blacktown, New South Wales 2148, Australia; and *C.S.I.R.O., Division of Mathematics and Statistics, P.O. Box 218, Lindfield, New South Wales 2070, Australia Summary. Two experiments were conducted to examine the effects of mouse epidermal growth factor (EGF) on the concentrations oftestosterone, LH and FSH injugular blood plasma and on the pituitary responsiveness to LHRH. In 20 rams treated with sub- cutaneous doses of EGF at rates of 85, 98 or 113 \g=m\g/kgfleece-free body weight, mean plasma LH and testosterone concentrations were significantly reduced (P < 0\m=.\05)at 6 h after treatment but not at 24 h. EGF treatment at 130 \g=m\g/kgfleece-free body weight suppressed the plasma content of these hormones for up to 48 h. Mean plasma FSH concentrations decreased significantly (P < 0\m=.\05)for up to 48 h after EGF treatment, the effect being most pronounced in rams with mean pretreatment FSH values >0\m=.\5ng/ml. Intravenous injections of 1\m=.\0 \g=m\gLHRH given to each of 5 rams before and at 6 h, 24 h and 72 h after EGF treatment produced LH and testosterone release patterns which paralleled those obtained in 5 control rams similarly treated with LHRH. These results suggest that, in rams, depilatory doses of mouse EGF temporarily impair gonadotrophin and androgen secretion by inhibiting LHRH release from the hypothalamus.
    [Show full text]
  • SNT, a Differentiation-Specific Target of Neurotrophic Factor-Induced Tyrosine Kinase Activity in Neurons and PC12 Cells STUART J
    MOLECULAR AND CELLULAR BIOLOGY, Apr. 1993, p. 2203-2213 Vol. 13, No. 4 0270-7306/93/042203-11$02.00/0 Copyright ) 1993, American Society for Microbiology SNT, a Differentiation-Specific Target of Neurotrophic Factor-Induced Tyrosine Kinase Activity in Neurons and PC12 Cells STUART J. RABIN, VAUGHN CLEGHON, ANtD DAVID R. KAPLAN* Eukaryotic Signal Transduction Group, Molecular Mechanisms of Carcinogenesis Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702-1201 Received 23 October 1992/Returned for modification 26 November 1992/Accepted 26 January 1993 To elucidate the signal transduction mechanisms used by ligands that induce differentiation and the cessation of cell division, we utilized p13 uc1agarose, a reagent that binds p34C2/cdk2 By using thi re t identified a 78- to 90-kDa species in PC12 pheochromocytoma cells that is rapidly phosphorylated on tyrosine following treatment with the differentiation factors nerve growth factor (NGF) and fibroblast growth factor but not by the mitogens epidermal growth factor or insulin. This species, called SNT (suc-associated neurotrophic factor-induced tyrosine-phosphorylated target), was also phosphorylated on tyrosine in primary rat cortical neurons treated with the neurotrophic factors neurotrophin-3, brain-derived neurotrophic factor, and fibroblast growth factor but not in those treated with epidermal growth factor. In neuronal and fibroblast cells, where NGF can also act as a mitogen, SNT was tyrosine phosphorylated to a much greater extent during NGF-induced differentiation than during NGF-induced proliferation. SNT was phosphorylated in vitro on serine, threonine, and tyrosine in pl3sucl-agarose precipitates from NGF-treated PC12 cells, indicating that this protein may be a substrate of kinase activities associated with p13suc1-p34cdc21cdk2 complexes.
    [Show full text]
  • Ontogenesis of Nerve Growth Factor
    Pediatr. Res. 16: 520-524 (1982) Ontogenesis of Nerve Growth Factor and Epidermal Growth Factor in Submaxillary Glands and Nerve Growth Factor in Brains of Immature Male Mice: Correlation with Ontogenesis of Serum Levels of Thyroid Hormones P. WALKER, M. E. WEICHSEL, JR.,'42' D. EVELETH, AND D. A. FISHER Department of Pediatrics, Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, California, USA Summary between 11 and 32 days of age. The known increase in mouse serum testosterone to adult male levels between 4-12 wk of age Using specific and sensitive radioimmunoassa~sfor may be a factor in the late increase in NGF and EGF elevations growth factor (NGF) and epidermal growth factor (EGF), we after 32 days of age. studied the developmental profde of NGF and EGF concentrations in male mouse submaxillary gland (SMG) from 2-60 days of age. We measured NGF concentrations in mouse cerebellum, cerebral Nerve growth factor (NGF) and epidermal growth factor (EGF) cortex, and brain stem from ages 2-32 days. In addition, we are found in highest concentrations in the submaxillary gland assessed mouse serum thyroxine (T4) and triiodothyronine (T3) (SMG) of the adult male mouse (13,22). Both proteins are found levels in SePerate groups of animals ranging from 5-50 days of in peritubular cells (32, 35) and are released concomitantly into age. Mean SMG EGF content and concentration exceeded that saliva (16,27). ~0thhave C-terminus arginine residues (2, 13) and of NGF between 2 and 18 days of age. At all subsequent ages, exist in SMG as high molecular weight complexes with specific however, mean NGF and EGF content and concentration were arginine esteropeptidase subunits (34).
    [Show full text]
  • Epidermal Growth Factor Suppresses Insulin-Like Growth Factor Binding
    (CANCER RESEARCH54, 3160-3166, June 15, 19941 Epidermal Growth Factor Suppresses Insulin-like Growth Factor Binding Protein 3 Levels in Human Papillomavirus Type 16-immortalized Cervical Epithelial Cells and Thereby Potentiates the Effects of Insulin-like Growth Factor 1' Joan R. Hembree,2Chapla Agarwal, and Richard L. Ecke& Departments of Physiology and Biophysics (J. R. H., C. A., R. L E.J, Demwtology [R. L. E.J, Reproductive Biology fR L. E.J, and Biochemistiy [J. R. H., R. L E.], Case Western Reserve University School ofMedicine, CIeveIand@Ohio 44106-4970 ABSTRACT growth. The types and quantities of IGFBPs produced are dependent on cell type and are influenced by a variety of hormones and growth Human ectocervical epithelial cells are a primary target for infection by factors (7—9).The effects of changes in IGFBP levels and/or type oncogemc papillomaviruses, which are strongly implicated as causative on IGF-I action are complex, in some cases potentiating (10—13) agents in the genesis of cervical cancer. Growth factors have been Impli and in others inhibiting (10, 14—16)IGF-I action. In the case of cated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human inhibition, it appears that soluble IGFBPs sequester IGFs and paplllomavirus (HPV) type 16-Immortalized eCtOCerVIcaIepithellal cell prevent their interaction with cell surface-associated IGFBPs lines to Investigate the effects of epidermal growth factor (EGF) and and/or IGF receptors (14—16). Potentiation of IGF-I action by insulin-like growth factor I (IGF-I) on cell proliferation and the produc IGFBP-3 has recently been attributed to association of IGFBP-3 tion of IGF binding proteins (IGFBPs).
    [Show full text]
  • Role of Epidermal Growth Factor in Carcinogenesis
    (CANCER RESEARCH 46, 1030-1037, March 1986] Role of Epidermal Growth Factor in Carcinogenesis Christa M. Stoscheck1 and Lloyd E. King, Jr.2 Veterans Administration Medical Center Research Service, Nashville 37203, and Department of Medicine, Division of Dermatology, Vanderbilt University Medical Center, Nashville, Tennessee 37232 Abstract rylates certain proteins on their tyrosine residues. Another mech anism may involve receptor clustering or a combination of clus For cell growth and division to occur, a large variety of meta tering and kinase activity which, in turn, produces an intracellular bolic processes must be carefully coordinated in the cell. Through signal, (b) Modulation of EGF-stimulated receptor activity (auto- evolutionary pressures, specific hormones and growth factors have acquired the ability to trigger a complex coordinated "pleio- regulation) occurs by at least three modes. First, binding and tropic growth response" in their target cells. This complex re kinase activities may be inhibited by phosphorylation of certain regulatory sites of the receptor. Second, receptor activity may sponse is mediated by specific cellular receptors and intracellular be reduced by receptor degradation following its interaction with messengers. Teleologically then, it makes sense that in onco- EGF. Third, sequestration of the EGF receptor in intracellular genesis this growth regulating network is utilized by the produc compartments may prevent EGF activation of this pool of recep tion of proteins which mimic growth factors, the activated form tors. of their receptors or, the messengers themselves. Several lines Several lines of evidence indicate that the EGF-stimulated cell of evidence indicate that the epidermal growth factor-stimulated regulatory system may play a role in carcinogenesis.
    [Show full text]