Mechanisms of Induced Cell Death in Bluetongue Virus Challenged Human Cell Lines
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Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 5-2009 Mechanisms of Induced Cell Death in Bluetongue Virus Challenged Human Cell Lines Justin Darrel Hoopes Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biology Commons Recommended Citation Hoopes, Justin Darrel, "Mechanisms of Induced Cell Death in Bluetongue Virus Challenged Human Cell Lines" (2009). All Graduate Theses and Dissertations. 252. https://digitalcommons.usu.edu/etd/252 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. MECHANISMS OF INDUCED CELL DEATH IN BLUETONGUE VIRUS CHALLENGED HUMAN CELL LINES by Justin Darrel Hoopes A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biology Approved: ___________________ ____________________ Joseph K.-K. Li Gregory J. Podgorski Major Professor Committee Member ___________________ ____________________ Dennis L. Welker Scott A. Ensign Committee Member Committee Member ___________________ ___________________ Jeffery R. Broadbent Byron R. Burnham Committee Member Dean of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2009 ii Copyright © Justin Darrel Hoopes 2008 All Rights Reserved iii ABSTRACT Mechanisms of Induced Cell Death in Bluetongue Virus Challenged Human Cell Lines by Justin Darrel Hoopes, Doctor of Philosophy Utah State University, 2009 Major Professor: Dr. Joseph K.-K. Li Department: Biology Bluetongue virus (BTV) is a pathogenic member of the Reoviridae family. BTV does not cause disease in humans, but is capable of selectively infecting and killing certain transformed human cell lines. Understanding BTV’s oncotrophism may lead to new therapeutics for treating cancer. This study focused on the underlying mechanisms of BTV-induced cell death in carcinoma cell lines. It was our hypothesis that BTV infects human carcinoma transformed cells, produces mRNA and protein, induces a strong inflammatory response, induces mitogen activated protein kinase (MAPK)-based pro-apoptotic signaling, inhibits PKB-based signaling, and eventually kills the cell by inducing apoptosis. Three carcinoma cell lines (A498, HEP-G2, and A549) were independently infected with BTV. In each cell line we determined: (1) cell viability over the course of infection; (2) BTV induced cytokine expression profile and magnitude of expression; (3) BTV viral RNA expression profile and magnitude of expression; iv (4) BTV viral protein expression profile and magnitude of expression; (5) changes in BTV induced cell death and cytokine expression in cells with protein kinase B (PKB), p38-MAPK, extracellular receptor kinase (ERK-1/2), stress-activated protein kinase (SAPK-JNK), Src kinase, platelet-derived growth factor receptor (PDGFR) kinase, epidermal growth factor receptor (EDGFR) kinase, or Janus kinase (JAK) activity inhibited; (6) intracellular changes in PKB, p38-MAPK, ERK- 1/2, and SAPK-JNK phosphorylation as a result of BTV infection; and (7) BTV- induced changes in tyrosine phosphorylation. We determined that BTV infects and kills all three cell lines in a cell line dependent manner. Relative cell death between cell lines was proportional to cytokine expression, but inversely proportional to viral protein expression. Only tyrosine kinase inhibitors influenced BTV-induced cell death and cytokine expression. Both A498 and A549 cells constitutively expressed phosphorylated PKB and p38 MAPK, of which both were de-phosphorylated during BTV infection. Tyrosine phosphorylation remained active, with elevated tyrosine phosphorylation exclusively in infected cells. We conclude that BTV-induced cell death and cytokine expression are a function of the cell’s response to infection and are directly related through intracellular signaling. These pathways are only partially poly I:C inducible, but include PKB and tyrosine kinase signaling. (171 pages) v ACKNOWLEDGMENTS I would like to thank Dr. Joseph K.-K. Li for his guidance and support over the years both academically and personally. I also thankfully acknowledge my committee members, Dr. Gregory J. Podgorski, Dr. Dennis L. Welker, Dr. Scott A. Ensign, and Dr. Jeffery R. Broadbent, for their patience, guidance, and academic support. I would also like to thank Quansys Biosciences for their contribution of resources, equipment, and expertise in cytokine testing as well as Ryan Jackson and Luck Peterson for their assistance in process and analyzing the RT-PCR data. I would like to particularly express gratitude to my wife, Kari Hoopes, for her continual patience and support through the years. I would not be the person I am without her. This research project was supported in part by Utah Agricultural Experiment Station Project #537 Justin Darrel Hoopes vi CONTENTS Page ABSTRACT ..........................................................................................................iii ACKNOWLEDGMENTS .......................................................................................vi LIST OF TABLES .................................................................................................ix LIST OF FIGURES ............................................................................................... x CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW.............................. 1 INTRODUCTION.................................................................. 1 OBJECTIVES AND HYPOTHESIS .................................... 30 REFERENCES.................................................................. .31 2. BLUETONGUE VIRUS (BTV)-INDUCED CELL DEATH CORRELATES DIRECTLY WITH BTV-INDUCED CYTOKINE EXPRESSION............................................................................... 51 ABSTRACT ........................................................................ 51 INTRODUCTION................................................................ 52 MATERIALS AND METHODS............................................ 54 RESLUTS........................................................................... 57 DISCUSSION ..................................................................... 61 REFERENCES................................................................... 71 3. BLUETONGUE VIRUS INDUCED CELL DEATH AND CYTOKINE EXPRESSION IS CAUSED BY THE CELL’S RESPONSE TO INFECTION................................................................................... 73 ABSTRACT ........................................................................ 73 INTRODUCTION................................................................ 74 MATERIALS AND METHODS............................................ 74 RESULTS........................................................................... 78 DISCUSSION ..................................................................... 94 REFERENCES................................................................... 98 vii 4. CELLULAR RESPONSES TO BLUETONGUE VIRUS INFECTION DO NOT INCLUDE ACTIVATION OF NFκB, P38 MAPK, OR JAK-STAT ........................................................... 99 ABSTRACT ........................................................................ 99 INTRODUCTION.............................................................. 100 MATERIALS AND METHODS.......................................... 101 RESULTS......................................................................... 107 DISCUSSION ................................................................... 132 REFERENCES................................................................. 148 5. SUMMARY AND CONCLUSION ................................................ 150 SUMMARY ....................................................................... 150 CONCLUSION ................................................................. 155 FUTURE RESEARCH...................................................... 156 CURRICULUM VITAE ...................................................................................... 157 viii LIST OF TABLES Table Page 1-1 Bluetongue Virus Gene Segments, Encoded Proteins, Location Within Virion, and Putative Function .......................................................... 6 2-1 Cytokine Expression (pg/mL) from A498, A549, and Vero Cells Seeded in 96 Well Plates 3 Days Post Treatment (n=4).......................... 59 3-1 Primer Sequences and Gene Location Used in the Quantitation of Each BTV cDNA by qRT-PCR ................................................................. 76 3-2 BHK-21 Cycle Threshold Values at 72 Hours P.I..................................... 90 3-3 A498 Cycle Threshold Values at 72 Hours P.I......................................... 91 3-4 HEP-G2 Cycle Threshold Values at 72 Hours P.I.................................... 91 3-5 A549 Cycle Threshold Values at 72 Hours P.I......................................... 92 ix LIST OF FIGURES Figure Page 1-1 A BTV-infected sheep from northern Europe. ............................................ 4 1-2 BTV virion structure ................................................................................... 6 1-3 Toll-like receptor (TLR) signaling ............................................................. 17 2-1 Mock-infected and BTV-infected HEP-G3 cells ....................................... 53 2-2 Mock-infected and BTV-infected A549