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Effect of Increased Hoxb4 Or Aml1-Eto on Human Megakaryocytic

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Effect of Increased Hoxb4 Or Aml1-Eto on Human Megakaryocytic EFFECT OF INCREASED HOXB4 OR AML1-ETO ON HUMAN MEGAKARYOCYTIC DEVELOPMENT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Yiming Zhong, M.S. Molecular Cellular and Developmental Biology Program The Ohio State University 2010 Dissertation Committee: Dr. Larry C. Lasky, Adviser Dr. James Waldman Dr. Joanne Trgovcich Dr. Xuefeng Bai ABSTRACT The transcription factors homeobox B4 (HoxB4) and acute myeloid leukemia-1 (AML1)-eight-twenty-one (ETO) have been shown to be important regulators in hematopoietic stem cells (HSCs) and in certain progenitor cells such as granulocyte-macrophage progenitors. However, their roles in megakaryopoiesis are unclear. In this study, we investigated the effect of increased HoxB4 or AML1-ETO on human megakaryocytic development and on in vitro platelet generation. We found that increased HoxB4 or AML1-ETO enhanced human early megakaryocytic development and that in vitro generation of platelets was increased by utilization of recombinant HoxB4 fusion protein and a three- dimensional (3D) culture system. Increased HoxB4 enhanced early megakaryocytic development in human erythro-megakaryocytic TF1 progenitor cell line and in CD34 positively-selected cord blood cells. Ectopic HoxB4 in human TF1 cells increased the antigen expression of two important megakaryocytic markers, CD61 and CD41a, increased the gene expression of thrombopoietin receptor (TpoR), stem cell leukemia-1 (Scl-1), Cyclin D1, Friend of GATA-1 (Fog-1) and Friend leukemia integration-1 (Fli-1) while it decreased the expression of cellular Myb (c-Myb) which contains sequences homologous to the avian myeloblastosis virus oncogene (v-myb). TpoR, cyclin D1, Fli-1 and Fog-1 are positive regulators of ii megakaryopoiesis and c-Myb is a potent negative modulator of megakaryopoiesis. HoxB4 RNA silencing in TF1 cells decreased the expression of CD61 and CD41a and decreased Fli-1 expression while it increased the expression of c-Myb. Recombinant triple protein transduction domain (tPTD)- HoxB4 fusion protein increased the percentages and absolute numbers of CD41a and CD61 positive cells during megakaryocytic differentiation of CD34 positively-selected cord blood cells and increased the numbers of colony-forming unit-megakaryocyte (CFU-Mk). Adding tPTD-HoxB4 fusion protein increased the gene expression of TpoR, Cyclin D1, Fog-1 and Fli-1 but inhibited c-Myb expression. In a four-stage 3D culture system, recombinant HoxB4 fusion protein was used to expand hematopoietic stem and progenitor cells in the first-two stages, and the expanded cells were induced for platelet differentiation in the last-two stages. We found that platelet production was increased in the 3D culture system with recombinant TAT-HoxB4 or tPTD-HoxB4 proteins added in the first two-stages of culture. The AML1-ETO fusion protein is generated by the t(8; 21) translocation and it is associated with about 40% of AML1 of M2 subtype. AML1 is a transcription factor belonging to the Runt-related transcription factor (RUNX) family and it involves in the development of normal hematopoiesis, for example, it is required for megakaryocytic maturation. AML1-ETO contains the DNA binding domain of AML1 but lacks the transcriptional activation domain of AML1 and it contains almost the entire ETO. So AML1-ETO functions as a dominant-negative factor of iii AML1 and represses transcription, and ETO domain contributes to this repression, which causes the development of AML. In this study, ectopic AML1- ETO was introduced into the human TF1 progenitor cells by lentivirus and its overexpression increased the expression of CD41a and CD61 in a dose- dependent manner. AML1-ETO overexpression in TF1 cells increased the gene expression of Fog-1 and decreased the c-Myb expression. Collectively, these data indicate that increased HoxB4 or AML1-ETO enhances human early megakaryocytic development by primarily regulating the gene expression of TpoR, Fog-1 and c-Myb and that in vitro generation of platelets is increased by utilization of recombinant HoxB4 proteins and 3D scaffolds. So manipulation of these transcription factors might be a way to produce platelets in vitro for use in transfusion medicine. iv DEDICATION Dedicated to my family v ACKNOWLEDGMENT I would like to thank my adviser, Dr. Larry C. Lasky, for his patience, support, advice and encouragement throughout my graduate education. I wish to express my appreciation to my dissertation committee members, Dr. James Waldman, Dr. Joanne Trgovcich and Dr. Xuefeng Bai, for their helpful advice and comments. I also thank my former advisers, Dr. Pan Zheng and Dr. Yang Liu, for their help. I would like to thank my colleagues, Brent Sullenbarger and Dr. Phil Wahr, for their assistance and cooperation. I also thank Dr. David Bisaro and Ms Jan Zinaich in the MCDB program for their help. I thank my family and friends for their continuous support, especially my wife Hongyan Liu and my two daughters for their love and support. vi VITA Nov. 13, 1976…………………......Born – Yueyang, China PR 1995 - 1999……………………..…B.S. Biology, Wuhan University 1999 – 2000………………............Department of Development, Red Heart King Group Co. Ltd 2000 – 2001……………….…….. Department of Scientific Research, Beijing Genomics Institute Chinese Academy of Sciences 2001 – 2004 ………………………M.S. Genetics, Institute of Genetics and Developmental Biology Chinese Academy of Sciences 2004 – Present……………..….….Graduate Research Associate The Ohio State University vii PUBLICATIONS 1. Yiming Zhong, Brent Sullenbarger, Larry C. Lasky. (2010) Effect of increased HoxB4 on human megakaryocytic development. Biochem Biophys Res Commun. 398(3):377-82. 2. ZHONG Yiming, JIANG Guanghuai, CHEN Xuewei, XIA Zhihui, LI Xiaobing, ZHU Lihuang, ZHAI Wenxue. (2003) Identification and gene prediction of a 24 kb region containing xa5, a recessive bacterial blight resistance gene in rice (Oryza sativa L.). CHINESE SCIENCE BULLETIN 48(24): 2725-2729 FIELDS OF STUDY Major Field: Molecular Cellular and Developmental Biology viii TABLE OF CONTENTS ABSTRACT .............................................................................................................. ii DEDICATION ........................................................................................................... v ACKNOWLEDGMENT ............................................................................................ vi VITA ......................................................................................................................... vii TABLE OF CONTENTS .......................................................................................... ix LIST OF TABLES ................................................................................................... xv LIST OF FIGURES .................................................................................................xvi ABREVIATIONS .....................................................................................................xix CHAPTER 1 ............................................................................................................. 1 General introduction ................................................................................................. 1 1.1 Hematopoietic stem/progenitor cells .......................................................... 1 1.2 Megakaryocytic development ..................................................................... 2 1.3 Regulation of megakaryocytic development .............................................. 3 1.4 Cell surface antigen expression during megakaryocytic development ..... 6 1.5 Human TF1 progenitor cell line .................................................................. 6 1.6 Cord blood................................................................................................... 7 ix 1.7 Significance and aims ................................................................................. 8 CHAPTER 2 ........................................................................................................... 15 Effect of increased HoxB4 on human megakaryocytic development ................... 15 2.1 Introduction ............................................................................................... 15 2.1.1 Roles of HoxB4 in hematopoiesis ..................................................... 15 2.1.2 In vitro expansion of HSCs can be achieved by HoxB4 overexpression or by directly adding recombinant HoxB4 protein................ 16 2.1.3 HoxB4 functions in a dose-dependent manner................................. 18 2.1.4 Molecular mechanisms of HoxB4’s effects on hematopoiesis ......... 19 2.1.5 Hypothesis.......................................................................................... 20 2.2 Materials and methods ................................................................................. 21 2.2.1 Cell culture ............................................................................................. 21 2.2.2 Lentiviral vectors .................................................................................... 22 2.2.3 Lentivirus production, concentration and titration ................................. 22 2.2.4 Recombinant HoxB4 protein ................................................................. 23 2.2.5 CFU and CFU-Mk assays ..................................................................... 23 2.2.6 Flow cytometry ......................................................................................
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