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E2f and Survivin – Key Players in Cellular Proliferation And E2F AND SURVIVIN – KEY PLAYERS IN CELLULAR PROLIFERATION AND TRANSFORMATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Baidehi Maiti, M.B.B.S. ***** The Ohio State University 2007 Dissertation Committee: Dr. Rachel A. Altura, Advisor Approved by Dr. Arthur H.M. Burghes Dr. Albert de la Chapelle Dr. Jiayuh Lin Advisor Graduate Program in Molecular Genetics ABSTRACT The E2F transcription factor family and survivin play a crucial role in maintaining homeostasis by coupling the vital processes of cell division and apoptosis. Deregulation of E2F or survivin signaling, lead to developmental defects in mice and are hallmarks of virtually every human cancer. Functionally, the mammalian E2F family consists of activators (E2F1-2 and E2F3a) and repressors (E2F3b and E2F4-8), which regulate cellular proliferation, apoptosis and differentiation. E2F7 and E2F8, which are the last two discovered mouse E2F family members, structurally form a sub-class within the repressor E2Fs. E2F7-8 are distinct in having a duplicated DNA binding domain, which is highly conserved across all the E2F family members. Both E2F7 and E2F8 inhibit cellular proliferation when overexpressed in mouse embryonic fibroblasts (MEFs). Expression of E2F7 and E2F8 are cell cycle regulated and are detected in the same adult mouse tissues suggesting a functional overlap or synergy. Consistent with the expanding complexity and functional diversity of the large mammalian E2F family, a cell cycle checkpoint is also regulated by the ii E2Fs. Simultaneous deletion of E2F1, E2F2 and E2F3, leads to a severe proliferative defect in the MEFs, correlating with the upregulation of p21 and the absence of cyclin dependent kinase activity. A proliferative defect is also observed in the MEFs conditionally deleted for survivin, a direct transcriptional target of the E2Fs. Survivin performs dual functions as a regulator of cell division and as an anti-apoptotic protein. During cell division, survivin ensures high fidelity chromosomal segregation and proper cytokinesis. Accordingly, survivin deleted MEFs fail to proliferate and become increasingly polyploid with an abnormal nuclear morphology. However, they do not undergo apoptosis, indicating a role of survivin in proliferation but not survival. Additionally, MEFs transformed with oncogenes c-Myc and H-RasV12 fail to proliferate following conditional deletion of survivin, demonstrating a requirement for survivin in the maintenance of transformation. Preliminary evidence also suggests that survivin is capable of collaborating with c-Myc or H- RasV12 in transforming MEFs, unraveling a novel role for survivin in cancer initiation. Collectively, these data uncover important roles of the E2F family and survivin in cellular proliferation and transformation. iii Dedicated to my parents, Dr. Bibekananda Maiti and Dr. Ilorasri Chakraborti for everything you do iv ACKNOWLEDGMENTS I would like to thank my advisor Dr. Rachel A. Altura for her excellent guidance, advice, encouragement and support. I am grateful to her for providing me a scientifically stimulating, enjoyable and productive mentor-student relationship. She has been instrumental in making this thesis possible. I wish to extend my gratitude to Dr. Gustavo W. Leone for giving me his time, supervision, scientific training and advice. I am thankful to all my current and past graduate advisory committee members, Dr. Arthur H. M. Burghes, Dr. Albert de la Chapelle, Dr. Jiayuh Lin, Dr. Lee F. Johnson and Dr. Michael C. Ostrowski for their time and effort towards my scientific development. I would like to acknowledge the contributions of all my present and past colleagues from the Altura and Leone labs. Thanks to Harold I. Saavedra, Rene Opavsky and Yuying Jiang, not only for their excellent scientific advice but also for being wonderfully supportive friends. It has also been a great learning experience to work with Cynthia McAllister, Michael P. Holloway, Lizhao Wu, Cynthia Timmers and Alain de bruin. v Thank you to Dr. Mark A. Seeger, Dr. Berl R. Oakley and Dr. David M. Bisaro for your kind and active support through some of the most challenging times of my career. I am also thankful to the staff of Molecular Genetics for their excellent timely help in the official procedures and paperwork. Finally, none of my achievements would be possible without the endless love and support of my family and friends. Thanks so much to my husband Sumit for remaining ever so loving, caring, understanding and supportive even when facing my displaced frustrations from failed experiments. Our little daughter Shivangi continues to be a source of enormous joy, love and energy for me. I am ever so thankful to my brother Baijayanta for his love, support and unwavering confidence in my abilities through rain or shine. My parents are a source of great inspiration for me. Their deepest love and devotion continues to brighten each day of my life. I can never thank them enough. vi VITA 1999……………………………….. M.B.B.S., Calcutta Medical College, University of Calcutta, Calcutta, India. 1999 - present……………………. Graduate Teaching and Research Associate, The Ohio State University, Columbus, Ohio. PUBLICATIONS Research Publication 1. Timmers C, Sharma N, Opavsky R, Maiti B, Wu L, Wu J, Orringer D, Trikha P, Saavedra HI, Leone G. E2f1, E2f2, and E2f3 control E2F target expression and cellular proliferation via a p53-dependent negative feedback loop. Mol Cell Biol. 2007 Jan;27(1):65-78. PMID: 17167174 2. Logan N, Graham A, Zhao X, Fisher R, Maiti B, Leone G, La Thangue NB. E2F-8: an E2F family member with a similar organization of DNA-binding domains to E2F-7. Oncogene. 2005 Jul 21;24(31):5000-4. PMID: 15897886 3. Maiti B, Li J, de Bruin A, Gordon F, Timmers C, Opavsky R, Patil K, Tuttle J, Cleghorn W, Leone G. Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation. J Biol Chem. 2005 May 6;280(18):18211-20. Epub 2005 Feb 18. PMID: 15722552 4. de Bruin A, Maiti B, Jakoi L, Timmers C, Buerki R, Leone G. Identification and characterization of E2F7, a novel mammalian E2F family member capable of blocking cellular proliferation. J Biol Chem. 2003 Oct 24;278(43):42041-9. Epub 2003 Jul 31. PMID: 12893818 5. Saavedra HI, Maiti B, Timmers C, Altura R, Tokuyama Y, Fukasawa K, Leone G. Inactivation of E2F3 results in centrosome amplification. Cancer Cell. 2003 Apr;3(4):333-46. PMID: 12726860 vii 6. Wu L, Timmers C, Maiti B, Saavedra HI, Sang L, Chong GT, Nuckolls F, Giangrande P, Wright FA, Field SJ, Greenberg ME, Orkin S, Nevins JR, Robinson ML, Leone G. The E2F1-3 transcription factors are essential for cellular proliferation. Nature. 2001 Nov 22;414(6862):457-62. PMID: 11719808 FIELDS OF STUDY Major Field: Molecular Genetics viii TABLE OF CONTENTS Page Abstract ........................................................................................................ii Dedication ....................................................................................................iv Acknowledgment ..........................................................................................v Vita ... ...........................................................................................................vii List of Tables ................................................................................................xi List of Figures...............................................................................................xii List of Abbreviations .....................................................................................xv Chapter 1: Introduction................................................................................1 E2F overview .....................................................................................2 The mammalian E2F transcription factor family.................................3 E2Fs regulate cellular proliferation ....................................................4 E2F in apoptosis ................................................................................5 E2Fs and cancer................................................................................6 Survivin overview...............................................................................7 Survivin as an anti-apoptotic protein..................................................8 Function of survivin in cell division.....................................................10 Survivin in development.....................................................................12 Survivin and cancer ...........................................................................13 Conclusion .........................................................................................15 Tables and figures .............................................................................16 Chapter 2: Cloning and characterization og mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation.......................25 ix Abstract..............................................................................................25 Introduction ........................................................................................26 Materials and Methods.......................................................................29 Results...............................................................................................36 Discussion .........................................................................................47
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