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Genomics-Based Characterization Of GENOMICS-BASED CHARACTERIZATION OF TUMOR SUPPRESSOR GENES IN THE CARDIOVASCULAR SYSTEM: A ROLE FOR ADENOMATOSIS POLYPOSIS COLI GENE IN HUMAN CARDIOVASCULAR DEVELOPMENT AND DISEASE Mojgan Rezvani A thesis submitted in conforrnity with the requirements for the degree of Doctor of Philosophy Graduate lnstitute of Medical Sciences University of Toronto 8 Copyright by Mojgan Rezvani (2001) 1 National Library Bibliotheque nationale du Canada Acquisitions and Acquisitions et Bibtiogtaphic Services services bibtiographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 OttawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. antorisation. GENOMICS-BASED CHARACTERIZATION OF TUMOR SUPPRESSOR GENES IN THE CARDIOVASCULAR SYSTEM: A ROLE FOR ADENOMATOSIS P9LYPOSIS COLI GENE IN HUMAN CARDIOVASCULAR DEVELOPMENT AND DISEASE Mojgan Rezvani, Ph.D. (2001) Graduate lnstitute of Medical Sciences University of Toronto CANADA ABST RACT Advances in sequence-based genome research using expressed sequence tag (EST) technology offers the opportunity for large-scale gene discovery. Complernenting the EST technology, the rapidly developing area of bioinformatics devoted to the collection, organization and analysis of DNA and protein sequences has advanced significantly our understanding of gene regulation during development and in disease states. The EST data can be analyzed in silico (cornputer based) to establish expression prof iles of cDNA libraries derived f rom specific tissues or organs. Expression profiles of different cDNA libraries can be compared by in silico Northern analysis as a tool to identify differentially expressed genes and to predict their functional roles in regulation of biological processes. In the current study, in silico Northem analysis of cardiac-derived EST's was employed to identify differentially expressed genes during cardiac development and in disease. Large scale expression profiling of EST's from cDNA libraries created from human fetal, normal adult and adult hypertrophie hearts revealed developrnentalfy and disease-dependent differentiat expression of severat genes involved in regulation of cell number and growth, including tumor suppressor and apoptosis-related genes, leading to the hypothesis that temporal changes in expression of these growth regulating genes may underlie developmental- and disease-specific alterations in cardiac growth phenotype. The tumor suppressor adenomatosis polyposis coli (APC) and its interacting protein P-catenin were found to be differentially expressed during cardiac development and in hypertrophic disease, resulting in higher abundance of APC in normal adult heart relative to hypertrophied and fetal heatts, and in reciprocal changes in p-catenin protein levels. Loss of function studies using antisense inhibition of APC translation in murine CzCi2 myoblasts inhibited cell proliferation and myotube formation and led to an increase in cell death, in parallel with an increase in basal p-catenin protein levels. Three novel APC protein isofoms were found to be expressed in the heart in a developmental- and disease-specific manner, and cornmensurate with this, expression levels of altematively spliced brain-specific (BS)- and exon-l containing APC gene isoforms were also found to Vary in developmental- and disease-specific manner. We conclude that APC plays a direct role in cardiac myocyte growth and differentiation and that differential and switching of altematively spliced and/or post-translationally modified APC isofoms may underlie, at least in part, some of the developmental- and disease- dependent alterations in cardiac growth phenotype. iii ACKNOWLEDGEMENTS 1 would like to thank my melitor, Dr. CC. Liew, for outstanding supervision through out the course of this degree, and Dr. M. Rabinovitch and Dr. Peter Liu for their excellent guidance and encouragement as members of my supervisory cornmittee. Thanks to IMS for their generous support of University of Toronto Open scholarship for the first year of my Ph.D. program as well as the yearly Merit Awards to date. Thanks to Heart and Stroke Foundation of Canada for awarding a research traineeship for the past four years. I would also like to thank my colleagues, David Hwang, Adam Dempsey, David Barrans, Ken Shaw, Christopher Ton and Dimitri Stamatiou and express my special gratitude to Eva Cukennan and Jack Liew for their helpful insights. Also. special thanks to Dr. V. Dzau and Dr. R. Pratts group, at the Brigham and women's hospital, Harvard Medical School in Boston for the opportunity of experiencing further scientific interaction as a exchange visiting scientist in the last year of my Ph.D. degree. I would like to specially thank Dr. L. G. Mello for his helpful comments and for sharing his extensive experience to prepare this thesis. I would like to specially thank my parents, Ahmad Rezvani and Fateme Haji and rny sisters Nooshin and Rashin for their unconditional love and support. Finally, I would like to express my eternal gratitude to the love of my life, rny husband, Günay Mete without whom none of this would have been possible. His undying support, inspiration, motivation and love have been the force to get me through it all. This Ph.D. degree belongs to hirn just as much as it belongs to me. TABLE Of CONTENTS Title Page i Abstract ii Acknowledgement iv Table of Contents v List of Abreviations viii List of Figures and tables ix CHAPTER 1: Introduction Preamble Hurnan Genome Project Expressed Sequence Tag (EST) Technology In Silico Northern Analysis EST Technology and Cardiovascular Genomics Genes lnvoived in Regulation of Growth in the Heart Tumor Suppressors, Cell Cycle and Cardiac Growth Suppressor Genes in Myocardium Tumor Suppressor APC in Cardiac Development and Disease A. Biochemistry and molecular biology of the APC gene B. Physiology and Pathophysiology of the APC gene Wingless 1 wnt Signaling Pathway p-Catenin Rationale, Hypothesis and Overview of Current Study A. Rationale B. Specific Hypotheses C. Experimental approach D. Significance CHAPTER 2: Apoptosis-related Genes Expressed in Cardiovascular ûevetoptnent and Disease: An EST Approach Abst ract lntroduction Genes lnvolved in Apoptosis - A Cardiovascular Perspective Interfeukin-converting enzyme (ICE) family: Caspases Bcl-2 Family Apoptosis-related Genes ldentified in the Cardiovascular System a) Effectors MA-3 Nip Family Stannin b) Suppressors DAD-1 Apoptosis Inhibifory Protein 47 c) Intemidiate Regulators 47 Tumor Necrosis Factor (TNF) and Fas Receptor Systems47 p38 Family Member p38-2G4 49 Apoptotic Genes Studied in Our Laboratory 49 Zinc Finger Proteins 49 ~53 50 APC 51 Summary and Future Directions 51 Acknowledgment 53 Refe rences 54 CHAPTER 3: Role of Adenornatous Polyposis Coli in Human Cardiac Development and Disease Abstract 63 Introduction 64 Experimental Procedures Total RNA extraction frorn Tissue and Cell Culture 67 cDNA Library Construction and LargeScale Sequencing 67 of cDNA Libraries Sequence and Digital Northern Analysis 67 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 68 Quantification of RT-PCR Results 69 lmmunoblotting Analysis 69 Ceil Culture 70 Antisense and Uptake Study 70 Cellular Proliferation Assay 71 Cellular Differentiation Assay 71 Statistics 72 Results Sequence and Computer-based Digital Northern Analysis 73 In Vitro Gene Expression Analysis (RT-PCR) 73 Protein Expression Level (Western Blot) 78 Cellular Growth and Differentiation Assay 78 Discussion 87 Acknowledgment 92 References 93 CHAPTER 4: Characterization of APC lsoforms in Caidiovascular System During Development and Disease Overview 97 Methods 1. Clones from human cDNA libraries 101 1 .l Isolation of cDNA clones cDNA clones 1 O1 1.2. Full Length AB1 Sequencing 1 O1 2. 5' Rapid Arnpllication of cDNA Ends (RACE) 101 2.t . RNA tsotation 2.2. Gene Specific Primer Design 2.3. PCR Amplification 2.4. Sequencing and Sequence Analysis of PCR Fragments 3. Sequencing of RT-PCR Products Arnplified with Prirnen Encompassing Exons 1 to 15 and Exon BS to 15 3.1 . Total RNA Extraction frorn Tissue 3.2. Primer Design 3.3. Reverse Transcription Polymerase Chain Reaction 3.4. Quantification of RT-PCR Results 3.5. Sequencing 3.6. Sequence Alignment and Analysis Results 1. Clones from human cDNA libraries 2. 5' RACE 2.1. PCR and Re-PCR 2.2. Sequencing of 5' RACE Products 2.3. Sequence Analysis 3. RT-PCR products amplified with primers spanning exons 1 to 15 and exons BS to 15 3.1. Differential Expression and Isofom Switching of APC in Myocardial Development and Disease 3.2. Sequence and BLAST Results Discussion CHAPTER 5: General Discussion, Conclusion and Future Directions 1. Synthesis of major findings 2. Surnmary 3. Conclusions 4. Overall significance of the findings 5. Future Directions REFERENCES
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