DOCSLIB.ORG

Effects of Engineered Costimulation on the Function of T Cell Subsets

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Effects of Engineered Costimulation on the Function of T Cell Subsets Effects of Engineered Costimulation on the Function of T Cell Subsets The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Boroughs, Angela C. 2019. Effects of Engineered Costimulation on the Function of T Cell Subsets. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41121316 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Effects of Engineered Costimulation on the Function of T Cell Subsets A dissertation presented by Angela Clare Boroughs to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Immunology Harvard University Cambridge, Massachusetts October 2018 © 2018 Angela Clare Boroughs All rights reserved. Dissertation Advisor: Dr. Marcela V. Maus Angela Clare Boroughs Effects of Engineered Costimulation on the Function of T Cell Subsets Abstract Progress in clinical adoptive immunotherapy was initially hindered by the cells’ lack of antigen specificity, poor engraftment, and limited persistence in the host. The introduction of chimeric antigen receptors (CARs) into T cells has overcome these obstacles by re-directing polyclonal T cells to a specific antigen with an extracellular binding domain and by including costimulation domains that enhance engraftment and persistence. From this observation, we hypothesized that the overexpression of CARs in different T cell subsets fundamentally changes their biology. CAR T cell products bearing either 4-1BB or CD28 costimulatory domains have been approved as therapies for leukemia and lymphoma. However, the engraftment kinetics, persistence, and toxicity profiles of CD28 versus 4-1BB CAR T cells are distinct. To obtain an in-depth understanding of the functional state of different types of CAR- modified T cells, we performed RNA sequencing on first- and second-generation CAR T cells both at rest and following CAR or endogenous T cell receptor (TCR) stimulation. We describe a high-resolution view of the transcriptional differences between 4-1BB and CD28 containing CAR T cells, including variances in cytokine profiles, cytokine receptors, and metabolic pathways. These transcriptional profiles define CAR signaling pathways that ultimately determine CAR T cell fate. iii Adoptive immunotherapy with regulatory T cells (Tregs) also holds promise in transplantation, graft-versus-host disease, and autoimmune diseases. Tregs are key modulators of inflammation and are important for peripheral tolerance. The challenges of using Tregs as adoptive immunotherapy mirror those for cancer: namely antigen specificity, engraftment, and persistence. We modified primary human Tregs with CARs bearing different costimulatory domains and performed rigorous analyses in vitro of the functional potential of Tregs. We showed that the presence of a CAR and the type of costimulation domain does not affect the Treg’s expression of Foxp3+. However, the costimulation domain does affect CAR-Treg cytokine production and surface marker expression. Furthermore 4-1BB costimulation decreases the suppressive function of CAR-Tregs. In vivo experiments, demonstrated that CAR-Tregs traffic to antigen expressing sites and can suppress antigen specific effector T cell responses. Our findings support the use of CD28-based CARs for tissue-specific suppression in the clinic. !! iv Acknowledgements I am deeply grateful for the opportunity to study in Harvard’s immunology program. Reflecting on my time here, I am ever more aware of how indebted I am, especially to the many educators who believed in my ability to succeed. I have learned that writing a thesis and performing experiments are not nearly as difficult as believing that I can successfully accomplish them. So, I cannot thank enough, my family, friends, and mentors who have supported and encouraged me to reach this goal. I would like to acknowledge the people who gave me precious first and second chances that were fundamental in shaping me as a student and scientist. Among those who built my confidence during my school years in South Africa, a few stand out. My great aunt Mary Hart spent many hours patiently guiding me through primary school homework. In 7th grade, my math teacher, Ms. Barrow, gave me a second chance to complete a test after I froze on the first try, not realizing what a turning point that would be for me. Two inspiring high school math teachers, Mrs. Rose Karam and Dr. Caroline Dickens, never doubted I could become an accomplished mathematician and scientist and gave me the ideal foundation to make that possible. My arrival at Harvard was first made possible by the encouragement of Dr. Fyodor Urnov, an adjunct professor at UC Berkeley, who during one brief semester as an undergraduate exchange student not only convinced me to switch fields from applied mathematics to molecular biology but also suggested that I apply to Harvard’s Immunology Program. Finally, to the immunology faculty who offered me a place in this program: I am eternally grateful that you took a chance on a “wild card” from South Africa. I would also like to acknowledge Dr. Ramnik Xavier for offering me a place in his lab, where I spent my first two years working with some of the most brilliant and inspirational scientists. I was fortunate to learn from Dr. Leigh Baxt, Dr. Nicole Desch, Dr. Dan Graham, Dr. Bernard Khor, Dr. Christine Becker, Jess Gracias, and Dr. Kara Conway. All of them helped me develop into a better scientist and have continued to be valuable sources of knowledge and dear friends since I moved on. With the never-failing support of Dr. Shiv Pillai, I began my PhD research anew in the Maus lab, yet another transformative second chance. I don’t have adequate words to express how grateful I am that Dr. Marcela Maus took me under her wing at that opportune moment. She had just opened her lab at Massachusetts General Hospital, and I doubt she had planned on braving a third-year graduate student so soon. Under her guidance, I found not only a knowledgeable and inspiring mentor, but also someone whose enthusiasm, patience, and encouragement boosted my confidence and my love of science. Despite her incredibly busy schedule, Dr. Maus has always been available to support me in whatever way I needed. She has kept me focused and provided v invaluable direction, while allowing me the flexibility to follow scientific questions in my own way. Every member of the Maus Lab, past and present, has helped me in some way, whether it be through critical thinking, technical assistance, or personal advice. I am thankful for their extraordinary friendship. I would especially like to thank Selena Lorrey, Lauren Riley, and Maria Cabral Rodriguez, the smart, enthusiastic, and oh-so-organized technicians who work tirelessly to keep the lab running smoothly. I was delighted when Rebecca Larson joined the lab as a fellow graduate student, brightening the lab with her enthusiasm and brilliance. The critical insights and advice on my research projects from my dissertation advisory committee, Dr. Laurence Turka, Dr. James Moon, and Dr. Shiv Pillai, have been invaluable. Some of my most treasured moments at Harvard were spent as a teaching fellow in the undergraduate immunology course. Again, I am grateful to Dr. Shiv Pillai for this opportunity and to all the students who helped me develop as an educator. My parents have been nothing but supportive, encouraging, and loving from before I can remember. From a young age, they instilled in me the wonders of learning, thinking, and teaching, not to mention the value of the arts and exercise that kept me somewhat balanced through my PhD. And I am thankful for the continuous encouragement from my diverse and incredible siblings, Meghan, Grace, and Patrick. I deeply miss my big South African family at home and thank them for the never-ending Snapchats and WhatsApp texts of encouragement that reminded me that there is life outside the lab. Throughout my time at Harvard, I have been inspired by those family members and friends who have so bravely lived through cancer and other serious illnesses. I am blessed to have angels and a dragonfly watching over me and inspiring me to appreciate each day and to keep persevering in a field that is making new cures possible. Finally, I am eternally grateful to my partner in dance and in life, my husband, Vaughan. He stuck with me through the two years we had to spend an ocean apart and then left his family and job in South Africa to restart in Massachusetts while I finished my degree. Throughout the highs and lows of this journey, Vaughan has been my rock, and I am grateful for the sacrifices he has made to be here for me while I reach toward my dream of a PhD in immunology. vi Dedication To Educators everywhere, who lift learners up, give second chances, and teach students to believe in themselves vii Table of Contents "##$%&'!(#!")*+)$$,$-!.('&+/012&+()!()!&3$!40)%&+()!(#!5!.$11!607'$&'!"""""""""""""""""""""""""""!#! $%&'()*'!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!###! $*+,-./012030,'&!""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""!4!
Load more

Recommended publications
  • Identification of Eqtls and Sqtls Associated with Meat Quality in Beef Joel D
    Leal-Gutiérrez et al. BMC Genomics (2020) 21:104 https://doi.org/10.1186/s12864-020-6520-5 RESEARCH ARTICLE Open Access Identification of eQTLs and sQTLs associated with meat quality in beef Joel D. Leal-Gutiérrez*, Mauricio A. Elzo and Raluca G. Mateescu Abstract Background: Transcription has a substantial genetic control and genetic dissection of gene expression could help us understand the genetic architecture of complex phenotypes such as meat quality in cattle. The objectives of the present research were: 1) to perform eQTL and sQTL mapping analyses for meat quality traits in longissimus dorsi muscle; 2) to uncover genes whose expression is influenced by local or distant genetic variation; 3) to identify expression and splicing hot spots; and 4) to uncover genomic regions affecting the expression of multiple genes. Results: Eighty steers were selected for phenotyping, genotyping and RNA-seq evaluation. A panel of traits related to meat quality was recorded in longissimus dorsi muscle. Information on 112,042 SNPs and expression data on 8588 autosomal genes and 87,770 exons from 8467 genes were included in an expression and splicing quantitative trait loci (QTL) mapping (eQTL and sQTL, respectively). A gene, exon and isoform differential expression analysis previously carried out in this population identified 1352 genes, referred to as DEG, as explaining part of the variability associated with meat quality traits. The eQTL and sQTL mapping was performed using a linear regression model in the R package Matrix eQTL. Genotype and year of birth were included as fixed effects, and population structure was accounted for by including as a covariate the first PC from a PCA analysis on genotypic data.
    [Show full text]
  • Downloaded the “Top Edge” Version
    bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Drosophila models of pathogenic copy-number variant genes show global and 2 non-neuronal defects during development 3 Short title: Non-neuronal defects of fly homologs of CNV genes 4 Tanzeen Yusuff1,4, Matthew Jensen1,4, Sneha Yennawar1,4, Lucilla Pizzo1, Siddharth 5 Karthikeyan1, Dagny J. Gould1, Avik Sarker1, Yurika Matsui1,2, Janani Iyer1, Zhi-Chun Lai1,2, 6 and Santhosh Girirajan1,3* 7 8 1. Department of Biochemistry and Molecular Biology, Pennsylvania State University, 9 University Park, PA 16802 10 2. Department of Biology, Pennsylvania State University, University Park, PA 16802 11 3. Department of Anthropology, Pennsylvania State University, University Park, PA 16802 12 4 contributed equally to work 13 14 *Correspondence: 15 Santhosh Girirajan, MBBS, PhD 16 205A Life Sciences Building 17 Pennsylvania State University 18 University Park, PA 16802 19 E-mail: [email protected] 20 Phone: 814-865-0674 21 1 bioRxiv preprint doi: https://doi.org/10.1101/855338; this version posted December 6, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 22 ABSTRACT 23 While rare pathogenic copy-number variants (CNVs) are associated with both neuronal and non- 24 neuronal phenotypes, functional studies evaluating these regions have focused on the molecular 25 basis of neuronal defects.
    [Show full text]
  • Literature Mining Sustains and Enhances Knowledge Discovery from Omic Studies
    LITERATURE MINING SUSTAINS AND ENHANCES KNOWLEDGE DISCOVERY FROM OMIC STUDIES by Rick Matthew Jordan B.S. Biology, University of Pittsburgh, 1996 M.S. Molecular Biology/Biotechnology, East Carolina University, 2001 M.S. Biomedical Informatics, University of Pittsburgh, 2005 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2016 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Rick Matthew Jordan It was defended on December 2, 2015 and approved by Shyam Visweswaran, M.D., Ph.D., Associate Professor Rebecca Jacobson, M.D., M.S., Professor Songjian Lu, Ph.D., Assistant Professor Dissertation Advisor: Vanathi Gopalakrishnan, Ph.D., Associate Professor ii Copyright © by Rick Matthew Jordan 2016 iii LITERATURE MINING SUSTAINS AND ENHANCES KNOWLEDGE DISCOVERY FROM OMIC STUDIES Rick Matthew Jordan, M.S. University of Pittsburgh, 2016 Genomic, proteomic and other experimentally generated data from studies of biological systems aiming to discover disease biomarkers are currently analyzed without sufficient supporting evidence from the literature due to complexities associated with automated processing. Extracting prior knowledge about markers associated with biological sample types and disease states from the literature is tedious, and little research has been performed to understand how to use this knowledge to inform the generation of classification models from ‘omic’ data. Using pathway analysis methods to better understand the underlying biology of complex diseases such as breast and lung cancers is state-of-the-art. However, the problem of how to combine literature- mining evidence with pathway analysis evidence is an open problem in biomedical informatics research.
    [Show full text]
  • Phosphatidylglycerol Incorporates Into Cardiolipin to Improve
    www.nature.com/scientificreports OPEN Phosphatidylglycerol Incorporates into Cardiolipin to Improve Mitochondrial Activity and Inhibits Received: 3 July 2017 Accepted: 7 March 2018 Infammation Published: xx xx xxxx Wei-Wei Chen1, Yu-Jen Chao1, Wan-Hsin Chang1, Jui-Fen Chan1 & Yuan-Hao Howard Hsu1,2 Chronic infammation and concomitant oxidative stress can induce mitochondrial dysfunction due to cardiolipin (CL) abnormalities in the mitochondrial inner membrane. To examine the responses of mitochondria to infammation, macrophage-like RAW264.7 cells were activated by Kdo2-Lipid A (KLA) in our infammation model, and then the mitochondrial CL profle, mitochondrial activity, and the mRNA expression of CL metabolism-related genes were examined. The results demonstrated that KLA activation caused CL desaturation and the partial loss of mitochondrial activity. KLA activation also induced the gene upregulation of cyclooxygenase (COX)-2 and phospholipid scramblase 3, and the gene downregulation of COX-1, lipoxygenase 5, and Δ-6 desaturase. We further examined the phophatidylglycerol (PG) inhibition efects on infammation. PG supplementation resulted in a 358- fold inhibition of COX-2 mRNA expression. PG(18:1)2 and PG(18:2)2 were incorporated into CLs to considerably alter the CL profle. The decreased CL and increased monolysocardiolipin (MLCL) quantity resulted in a reduced CL/MLCL ratio. KLA-activated macrophages responded diferentially to PG(18:1)2 and PG(18:2)2 supplementation. Specifcally, PG(18:1)2 induced less changes in the CL/MLCL ratio than did PG(18:2)2, which resulted in a 50% reduction in the CL/MLCL ratio. However, both PG types rescued 20–30% of the mitochondrial activity that had been afected by KLA activation.
    [Show full text]
  • Role and Regulation of Snon/Skil and PLSCR1 Located at 3Q26.2
    University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 9-18-2014 Role and Regulation of SnoN/SkiL and PLSCR1 Located at 3q26.2 and 3q23, Respectively, in Ovarian Cancer Pathophysiology Madhav Karthik Kodigepalli University of South Florida, [email protected] Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the Cell Biology Commons, Microbiology Commons, and the Molecular Biology Commons Scholar Commons Citation Kodigepalli, Madhav Karthik, "Role and Regulation of SnoN/SkiL and PLSCR1 Located at 3q26.2 and 3q23, Respectively, in Ovarian Cancer Pathophysiology" (2014). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/5426 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Role and Regulation of SnoN/SkiL and PLSCR1 Located at 3q26.2 and 3q23, Respectively, in Ovarian Cancer Pathophysiology by Madhav Karthik Kodigepalli A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cell and Molecular Biology Department of Cell Biology, Microbiology and Molecular Biology College of Arts and Sciences University of South Florida Major Professor: Meera Nanjundan, Ph.D. Richard Pollenz, Ph.D. Patrick Bradshaw, Ph.D. Sandy Westerheide, Ph.D. Date of Approval: September 18, 2014 Keywords: Chemotherapeutics, phospholipid scramblase, toll-like receptor, interferon, dsDNA Copyright © 2014, Madhav Karthik Kodigepalli Dedication I dedicate this research at the lotus feet of Bhagwan Sri Sathya Sai Baba and all the Masters for I am what I am due to their divine grace.
    [Show full text]
  • Phosphatidylserine Exposure Controls Viral Innate Immune Responses by Microglia
    Article Phosphatidylserine Exposure Controls Viral Innate Immune Responses by Microglia Highlights Authors d Phosphatidylserine exposure tags virally transduced cells for Yusuf Tufail, Daniela Cook, microglial phagocytosis Lawrence Fourgeaud, ..., Clodagh C. O’Shea, Greg Lemke, d Engulfment by microglia is TAM receptor dependent Axel Nimmerjahn d Inhibition of phospholipid scramblase 1 activity protects from phagocytosis Correspondence [email protected] d Rescued cells remain viable allowing prolonged transgene expression In Brief Tufail et al. show that inhibiting phospholipid scramblase 1 activity in virally transduced cells prevents intracellular calcium dysregulation, phosphatidylserine externalization, and TAM receptor-dependent microglial phagocytosis, providing a novel mechanism through which innate immune responses to viral vectors may be controlled. Tufail et al., 2017, Neuron 93, 574–586 February 8, 2017 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.neuron.2016.12.021 Neuron Article Phosphatidylserine Exposure Controls Viral Innate Immune Responses by Microglia Yusuf Tufail,1 Daniela Cook,1 Lawrence Fourgeaud,2 Colin J. Powers,3 Katharina Merten,1 Charles L. Clark,1 Elizabeth Hoffman,1 Alexander Ngo,1 Kohei J. Sekiguchi,1 Clodagh C. O’Shea,3 Greg Lemke,2,4 and Axel Nimmerjahn1,5,* 1Waitt Advanced Biophotonics Center 2Molecular Neurobiology Laboratory 3Molecular and Cell Biology Laboratory 4Immunobiology and Microbial Pathogenesis Laboratory The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA 5Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2016.12.021 SUMMARY spread. Under certain conditions, maladaptive innate immune responses can lead to undesired cell loss through microglial Microglia are the intrinsic immune sentinels of the phagocytosis (Brown and Neher, 2014).
    [Show full text]
  • Integrated Transcriptomic and Neuroimaging Brain Model Decodes
    RESEARCH ARTICLE Integrated transcriptomic and neuroimaging brain model decodes biological mechanisms in aging and Alzheimer’s disease Quadri Adewale1,2,3, Ahmed F Khan1,2,3, Felix Carbonell4, Yasser Iturria-Medina1,2,3*, Alzheimer’s Disease Neuroimaging Initiative† 1Neurology and Neurosurgery Department, Montreal Neurological Institute, McGill University, Montreal, Canada; 2McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Canada; 3Ludmer Centre for Neuroinformatics and Mental Health, McGill University, Montreal, Canada; *For correspondence: 4Biospective Inc, Montreal, Canada [email protected] †Data used in preparation of this article were partly obtained from the Alzheimer’s Disease Abstract Both healthy aging and Alzheimer’s disease (AD) are characterized by concurrent Neuroimaging Initiative (ADNI) alterations in several biological factors. However, generative brain models of aging and AD are database (adni.loni.usc.edu). As limited in incorporating the measures of these biological factors at different spatial resolutions. such, the investigators within the Here, we propose a personalized bottom-up spatiotemporal brain model that accounts for the ADNI contributed to the design direct interplay between hundreds of RNA transcripts and multiple macroscopic neuroimaging and implementation of ADNI modalities (PET, MRI). In normal elderly and AD participants, the model identifies top genes and/or provided data but did not modulating tau and amyloid-b burdens, vascular flow, glucose metabolism, functional activity, and participate in analysis or writing atrophy to drive cognitive decline. The results also revealed that AD and healthy aging share of this report. A complete listing specific biological mechanisms, even though AD is a separate entity with considerably more altered of ADNI investigators can be pathways.
    [Show full text]
  • PLSCR3 Polyclonal Antibody Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP58592
    苏州工业园区双圩路9号1幢 邮 编 ： 215000 电 话 ： 0512-88856768 PLSCR3 Polyclonal Antibody Purified Rabbit Polyclonal Antibody (Pab) Catalog # AP58592 Specification PLSCR3 Polyclonal Antibody - Product info Application IHC-P, IHC-F, IF Primary Accession Q9NRY6 Reactivity Rat, Pig, Cow Host Rabbit Clonality Polyclonal Calculated MW 31648 PLSCR3 Polyclonal Antibody - Additional info Gene ID 57048 Other Names Phospholipid scramblase 3, PL scramblase 3, Ca(2+)-dependent phospholipid scramblase 3, PLSCR3 Dilution Elisa=1:500-1000,IHC-P=1:100-500,IHC-F=1:100-500,IF=1:100-500, Format 0.01M TBS(pH7.4) with 1% BSA, 0.09% (W/V) sodium azide and 50% Glyce Storage Store at -20 °C for one year. Avoid repeated freeze/thaw cycles. When reconstituted in sterile pH 7.4 0.01M PBS or diluent of antibody the antibody is stable for at least two weeks at 2-4 °C. PLSCR3 Polyclonal Antibody - Protein Information Name PLSCR3 Function Catalyzes calcium-induced ATP-independent rapid bidirectional and non-specific movement of the phospholipids (lipid scrambling or lipid flip-flop) between the inner and outer membrane of the mitochondria (PubMed:<a href="http://www.uniprot.org/citations/14573790" target="_blank">14573790</a>, PubMed:<a href="http://www.uniprot.org/citations/17226776" target="_blank">17226776</a>, PubMed:<a href="http://www.uniprot.org/citations/18358005" target="_blank">18358005</a>, PubMed:<a href="http://www.uniprot.org/citations/29337693" target="_blank">29337693</a>, PubMed:<a href="http://www.uniprot.org/citations/31769662" target="_blank">31769662</a>). Plays an important role in mitochondrial respiratory function, morphology, and apoptotic response (PubMed:<a href="http://www.uniprot.org/citations/14573790" target="_blank">14573790</a>, PubMed:<a href="http://www.uniprot.org/citations/17226776" target="_blank">17226776</a>, PubMed:<a href="http://www.uniprot.org/citations/18358005" target="_blank">18358005</a>, PubMed:<a href="http://www.uniprot.org/citations/12649167" target="_blank">12649167</a>).
    [Show full text]
  • ALG-2-Interacting Tubby-Like Protein Superfamily Member PLSCR3 Is Secreted by an Exosomal Pathway and Taken up by Recipient Cultured Cells
    Biosci. Rep. (2013) / 33 / art:e00026 / doi 10.1042/BSR20120123 ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells Tatsutoshi INUZUKA*, Akira INOKAWA*, Cen CHEN*, Kumiko KIZU†, Hiroshi NARITA†, Hideki SHIBATA* and Masatoshi MAKI*1 *Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, and †Department of Food and Nutrition, Kyoto Women’s University, 35 Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605-8501, Japan Synopsis PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activit- ies observed in vitro. Using a previously established cell line of HEK-293 (human embryonic kidney-293) cells con- stitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 (apoptosis-linked gene 2) Ca2 + -dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-BP (2-bromopalmitate, a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich re- gion were necessary for efficient secretion, but ABSs (ALG-2-binding sites) were dispensable. Overexpression of GFP (green fluorescent protein)-fused VPS4BE235Q, a dominant negative mutant of an AAA (ATPase associated with various cellular activities) ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localized to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP–Rab5AQ79L).
    [Show full text]
  • 1P31, 7Q21 and 18Q21 Chromosomal Aberrations and Candidate Genes in Acquired Vinblastine Resistance of Human Cervical Carcinoma KB Cells
    1155-1164 4/4/08 16:56 Page 1155 ONCOLOGY REPORTS 19: 1155-1164, 2008 1p31, 7q21 and 18q21 chromosomal aberrations and candidate genes in acquired vinblastine resistance of human cervical carcinoma KB cells JIN WANG1,3, LAI-SHAN TAI2, CHI-HUNG TZANG1, WAN FONG FONG1, XIN-YUAN GUAN2 and MENGSU YANG1 1Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon; 2Department of Clinical Oncology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, P.R. China Received January 25, 2008; Accepted March 3, 2008 Abstract. Vinblastine (VBL) is used to treat certain kinds of resistance. This study also demonstrates that the combination cancer including Hodgkin's lymphoma, lung cancer, breast of CGH and cDNA microarray is a very useful tool to detect cancer, testicular cancer and cervical carcinoma. However, drug resistant targets in cancer treatment. the rapid development of resistance during therapy remains a major clinical challenge. In order to reverse cancer cell Introduction resistance, the goal of this study was to find differentially expressed genes and chromosomal alterations in multidrug Drug resistance is the major obstacle to be overcome during resistant (MDR) KB-v1 cells, further to probe the relation- the systemic therapy of cancer. The terms of drug resistance ship between drug resistance and differential genes, and and sensitivity are relative conditions that must be defined chromosomal changes in MDR cancer cells. Comparative with respect to some standard reference frames. Once a drug genomic hybridization (CGH) analysis of MDR KB-v1 and has achieved a critical threshold, it will interact with a range their parental KB-3-1 cells revealed chromosomal changes; of cellular macromolecules.
    [Show full text]
  • Genome-Wide Survey of Microrna–Transcription Factor Feed-Forward Regulatory Circuits in Humanw
    View Article Online / Journal Homepage / Table of Contents for this issue PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems Genome-wide survey of microRNA–transcription factor feed-forward regulatory circuits in humanw Angela Re,za Davide Cora´,zbd Daniela Tavernacd and Michele Caselle*bd Received 7th January 2009, Accepted 27th April 2009 First published as an Advance Article on the web 19th June 2009 DOI: 10.1039/b900177h In this work, we describe a computational framework for the genome-wide identification and characterization of mixed transcriptional/post-transcriptional regulatory circuits in humans. We concentrated in particular on feed-forward loops (FFL), in which a master transcription factor regulates a microRNA, and together with it, a set of joint target protein coding genes. The circuits were assembled with a two step procedure. We first constructed separately the transcriptional and post-transcriptional components of the human regulatory network by looking for conserved over-represented motifs in human and mouse promoters, and 30-UTRs. Then, we combined the two subnetworks looking for mixed feed-forward regulatory interactions, finding a total of 638 putative (merged) FFLs. In order to investigate their biological relevance, we filtered these circuits using three selection criteria: (I) GeneOntology enrichment among the joint targets of the FFL, (II) independent computational evidence for the regulatory interactions of the FFL, extracted from external databases, and (III) relevance of the FFL in cancer. Most of the
    [Show full text]
  • The Ins and Outs of Phospholipid Asymmetry in the Plasma Membrane: Roles in Health and Disease
    Critical Reviews in Biochemistry and Molecular Biology, 2009; 44(5): 264–277 REVIEW ARTICLE The ins and outs of phospholipid asymmetry in the plasma membrane: roles in health and disease Bengt Fadeel1, and Ding Xue2 1Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden, and 2Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado, USA Abstract A common feature of all eukaryotic membranes is the non-random distribution of different lipid species in the lipid bilayer (lipid asymmetry). Lipid asymmetry provides the two sides of the plasma membrane with different biophysical properties and influences numerous cellular functions. Alteration of lipid asym- metry plays a prominent role during cell fusion, activation of the coagulation cascade, and recognition and removal of apoptotic cell corpses by macrophages (programmed cell clearance). Here we discuss the origin and maintenance of phospholipid asymmetry, based on recent studies in mammalian systems as well as in Caenhorhabditis elegans and other model organisms, along with emerging evidence for a conserved role of mitochondria in the loss of lipid asymmetry during apoptosis. The functional significance of lipid asym- metry and its disruption during health and disease is also discussed. Keywords: Phospholipid asymmetry; Caenhorhabditis elegans; programmed cell clearance; mitochondria; phosphatidylserine; phospholipid scramblases; aminophospholipid translocases For personal use only.
    [Show full text]