Marshall University Marshall Digital Scholar Theses, Dissertations and Capstones 1-1-2011 Identification of Genes Involved in Hematopoietic Stem Cell Differentiation and Leukemia Differentiation and Leukemia Jasjeet Bhullar [email protected] Follow this and additional works at: http://mds.marshall.edu/etd Part of the Hematology Commons, and the Medical Cell Biology Commons Recommended Citation Bhullar, Jasjeet, "Identification of Genes Involved in Hematopoietic Stem Cell Differentiation and Leukemia Differentiation and Leukemia" (2011). Theses, Dissertations and Capstones. Paper 23. This Dissertation is brought to you for free and open access by Marshall Digital Scholar. It has been accepted for inclusion in Theses, Dissertations and Capstones by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected]. Identification of genes involved in hematopoietic stem cell differentiation and leukemia Dissertation submitted to the Graduate College of Marshall University in partial fulfillment of the requirement for the degree of Doctor of Philosophy by Jasjeet Bhullar Approved by: Dr. Vincent E Sollars, Ph.D., Committee Chairperson Dr. Richard Niles, Ph.D. Dr. Todd Green, Ph.D. Dr. Beverly Delidow, Ph.D. Dr. Susan Jackman, Ph.D. Department of Biochemistry and Microbiology, Marshall University Joan C. Edwards School of Medicine Huntington, West Virginia April 2011 ABSTRACT Hematopoiesis is maintained by a proper balance between self renewal and multipotent differentiation of the hematopoietic stem cells (HSC). Acute myelogenous leukemia (AML) is characterized by the blockage in the differentiation of HSC, while self renewal and proliferation is preserved. It is important to understand the mechanisms involved in the inhibition of hematopoietic differentiation and maintenance of the HSC state in order to develop better therapies for AML. In these studies I have explored the role of Hsp90, omega-3 fatty acids and YB-1 in hematopoietic differentiation. EML, a hematopoietic precursor cell line, was used as a model for the hematopoietic system in these studies. My preliminary data showed the activation of Wnt signaling upon inhibition of Hsp90 in EML cells. This data suggested the involvement of Hsp90 in the regulation of Wnt signaling in EML cells. Moreover, my initial data with fatty acid studies indicated that omega-3 fatty acids could affect Wnt signaling in EML cells. Unfortunately, further progression of both these studies was marred by variability in my data. In my latest study, I have identified YB-1 as a marker involved in the maintenance of the hematopoietic stem cell state. YB-1 was found to be highly expressed in the EML cell line and in the mouse bone marrow-derived HSC and myeloid progenitor cells. In addition, YB-1 expression was downregulated during myeloid differentiation in retinoic acid (RA) and granulocyte macrophage colony stimulating factor (GM-CSF) treated EML cells, as well as in the granulocytes derived from mouse bone marrow. Further, abnormal YB-1 expression was observed in myeloid leukemic cell lines. Knockdown of YB-1 expression and arsenic trioxide treatment (As 2O3) in erythroleukemic, K562 cell line resulted in apoptosis and inhibition of cell proliferation. Most importantly, these treatments led to the induction of megakaryocytic differentiation in these cells. Overall my data suggests that increased expression of YB-1 in the leukemic cells contributes to the leukemic cell properties by promoting cell proliferation, cell survival and blocking cell differentiation. Thus, YB-1 could be a potential target for therapy in myeloid leukemia. ii DEDICATION I would like to dedicate this work to my parents, Satpal and Jasmeet Bhullar. Without their knowledge, wisdom, and guidance, I would not have the goals I have; to strive and be the best to reach my dreams. iii ACKNOWLEDGEMENTS I would like to thank my family for being there for me when I needed them the most. It is due to their encouragement, support and love I was able to come so far to pursue my dream. I would like to sincerely thank my brother, Simarjeet Bhullar who was responsible for giving a direction to my life. I have always admired his courage and determination and followed in his footsteps. I love my parents and I sincerely appreciate their patience and support. I can not thank enough my husband, Dr. Sandeep Joshi, with whose support I was able to achieve my goal of graduate education. He motivated, encouraged and guided me through this. He took care of our son while I was working on my dissertation. I would like to thank almighty god for giving me his blessings and a wonderful son Prateik. I would also like to thank my mother- and father-in-law who took care of things at home while I was writing. I would like to thank all my labmates for some of the quality time we spent during the course of this journey particularly Dr. Jennifer Napper, Dr. Mindy Varney, Harsh Pratap, Ayah Arafa, James Buchanan and Jonathan Lewis. I would thank the BMS program for giving me this opportunity to fulfill my goal of graduate education. Also, I’d like to thank all my BMS friends who made me cherish my graduate school experience. I am going to miss all of you. I would like to thank my advisor, Dr. Sollars for guiding me through this work. His patience and support is appreciated. I would thank my committee: Dr. Susan Jackman, Dr. Beverely Delidow, Dr. Richard Niles and Dr. Green who helped me become a better scientist. It’s through their guidance I strived hard to learn more. iv TABLE OF CONTENTS TITLE PAGE ................................................................................................................................. i ABSTRACT ................................................................................................................................... ii DEDICATION PAGE ................................................................................................................. iii ACKNOWLEDGEMENTS ........................................................................................................ iv TABLE OF CONTENTS ..............................................................................................................v LIST OF FIGURES .................................................................................................................... vii LIST OF ABBREVIATIONS .......................................................................................................x CHAPTER ONE: INTRODUCTION ..........................................................................................1 Hematopoiesis ...........................................................................................................................1 Acute Myelogenous Leukemia ................................................................................................4 Stress response .........................................................................................................................6 Hsp90 isoforms and structure .................................................................................................8 Role of Hsp90 in cancer .........................................................................................................12 Hsp90 inhibitors ....................................................................................................................13 Y Box Protein (YB-1) .............................................................................................................15 YB-1 expression, structure and function .............................................................................16 Biological role of YB-1 ...........................................................................................................19 Role of YB-1 in cancer ...........................................................................................................20 Role of YB-1 in hematological malignancies .......................................................................22 Wnt signalling ........................................................................................................................24 Wnt signalling and hematopoietic stem cells .......................................................................26 Omega fatty acids and cancer ...............................................................................................28 CHAPTER TWO: YB-1 expression and function in early hematopoiesis and leukemia ....32 ABSTRACT ............................................................................................................................33 INTRODUCTION..................................................................................................................34 MATERIALS AND METHODS ..........................................................................................36 RESULTS ...............................................................................................................................42 v DISCUSSION .........................................................................................................................54 CHAPTER THREE: Activation of the canonical Wnt signaling in hematopoietic precursor, EML cell line upon Hsp90 inhibition ......................................................................60 ABSTRACT ............................................................................................................................60 INTRODUCTION..................................................................................................................61