UGENE, A NEWLY IDENTIFIED PROTEIN THAT IS COMMONLY OVER- EXPRESSED IN CANCER, AND THAT BINDS URACIL DNA-GLYCOSYLASE by CHUNGUANG GUO Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Sanford Markowitz Department of Molecular Biology and Microbiology CASE WESTERN RESERVE UNIVERSITY January, 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Chunguang Guo candidate for the Ph.D. degree *. (signed) Robert Silverman (chair of the committee) George Stark Andrei Gudkov Sanford D Markowitz (date) 06/24/08 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Table of contents List of tables .............................................................................................................4 List of figures ...........................................................................................................5 Acknowledgement ...................................................................................................7 Abstract .............................................................................................................9 Chapter 1 Introduction .......................................................................................10 1.1 Epidemiology of Colorectal Cancer .............................................................10 1.2 Staging, Therapy and Prognosis ...................................................................10 1.3 Genetics of Colon Cancer .............................................................................11 1.4 The Role of TGF-β Signaling in Colorectal Cancer .....................................13 1.4.1 TGFbR2 ..................................................................................................15 1.4.2 Smads .....................................................................................................17 1.5 DNA repair pathways and colorectal cancer ................................................18 1.6 Microarray techniques in cancer research ....................................................21 Chapter 2 Identification of a novel gene, Ugene, over-expressed in most cancer types ..............................................................................24 2.1 Expression of EST U46258 transcript is consistently up- regulated in colon cancer by DNA microarray analysis ..............................24 2.2 Identification of Ugene ................................................................................26 2.3 Validation of Ugene expression in colon cancer vs. normal colon ............................................................................................................31 2.3.1 Ugene expression is elevated in colon cancer ......................................31 2.3.2 Both Ugene-p and Ugene-q are over-expressed in colon cancer .........34 1 2.3.3 Ugene is over-expressed in many types of cancers ...............................35 2.4 Investigation of the possible causes for the elevated expression level of Ugene in colon cancers ..................................................................36 2.4.1 Chromosome amplification and rearrangement ...................................36 2.4.2 TGF-β regulation ..................................................................................38 2.5 Ugene expression is cell cycle denpendent .................................................41 2.6 Ugene encodes a nuclear protein .................................................................44 Materials and methods ........................................................................................46 Chapter 3 Epitope tagging of endogenous Ugene ..............................................52 Materials and methods ........................................................................................59 Chapter 4 Investigation of potential roles of Ugene in cell proliferation and oncogenesis ............................................................64 4.1 Potential roles of Ugene in cell transformation ............................................64 4.1.1 NIH 3T3 cells transformation ...............................................................64 4.1.2 REF-52 cells transformation .................................................................66 4.1.3 WI-38 cells transformation ...................................................................67 4.1.4 HME1 cells transformation ...................................................................69 4.2 Potential roles of Ugene in cell proliferation ...............................................70 4.3 Ugene potential interaction partners .............................................................73 4.3.1 Identification of Ugene potential partners ............................................73 4.3.2 Validation of Ugene potential partners .................................................74 4.3.3 Ugene-p binds to the NH2-terminus of UNG2 ........................................81 2 4.3.4 Ugene binding does not directly alter UNG2 enzymatic activity or localization ......................................................................................................84 Materials and methods ........................................................................................90 Chapter 5 Discussion and Future Directions .....................................................95 Bibliography ..........................................................................................................98 3 List of tables Table 1-1 DNA repair genes linked to familial cancer syndromes……….……19 Table 2-1 Difference between Ugene-p and Ugene-q…………………….……31 Table 2-2 Ratio of Ugene-p and Ugene-q in colon cancer cell lines……………34 Table 2-3 Ratio of Ugene-p and Ugene-q in V330 ……………………….……41 4 List of figures Figure 1-1 Genetic changes associated with colorectal ........................................12 Figure 2-1 Expression of EST U46258 on DNA microarrays ..............................26 Figure 2-2 Structure of the Ugene locu.................................................................29 Figure 2-3 Ugene consists of two different transcripts, Ugene-p and Ugene-q ....31 Figure 2-4 Ugene mRNA expression in normal and cancer samples ...................34 Figure 2-5 Ugene expression in cancers ...............................................................36 Figure 2-6 Detection of amplification, rearrangement of Ugene ..........................37 Figure 2-7 TGF-β regulation of Ugene expression on microarray .......................39 Figure 2-8 Ugene can be down-regulated by TGF-β ............................................40 Figure 2-9 Cell cycle profiling of Ugene mRNA expression ...............................43 Figure 2-10 Subcellular localization of Ugene .....................................................45 Figure 3-1 Schematic diagram of tagging endogenous protein with 3 Flag .......54 Figure 3-2 3xFlag epitope tagging of endogenous Ugene-p .................................57 Figure 3-3 Timelines for 3xFlag knock-in and antibody production ....................58 Figure 4-1 NIH 3T3 cell transformation assay .....................................................66 Figure 4-2 Anchorage independent growth assay of WI-38 cells .........................69 Figure 4-3 Suppression of Ugene expression by specific siRNAs .......................71 Figure 4-4 Colony formation assays after siRNA transfection .............................73 Figure 4-5 Flag pull-down of Ugene-p .................................................................74 Figure 4-6 Protein-protein interaction of Ugene-p and UNG2 .............................77 Figure 4-7 Ugene-q does not interact with UNG2 ................................................78 Figure 4-8 Protein-protein interaction of Ugene and NOSIP ...............................80 5 Figure 4-9 NOSIP doesn’t form complex with UNG2 .........................................81 Figure 4-10 Mapping of the UNG2 domain for binding to Ugene-p ....................84 Figure 4-11 Western analysis of UNG in UNG1 null and UNG null cells ...........87 Figure 4-12 Assays of UNG enzymatic activity ...................................................88 Figure 4-13 UNG2 localization in DLD1 cells with and without presence of Ugene-p .............................................................................................89 6 Acknowledgement First, I would like to express my sincere gratitude to my thesis advisor, Dr. Sanford D Markowitz, not only for his great guidance and endless support in my project, but also for his patience and encouragement throughout the course of my graduate studies. In past six years, he has taught me innumerable lessons and insights to think critically, to experimentally query the unknown, and to write about science. His support has meant more to me than I can express here, and he has empowered me with the strong motivation and unbendable perseverance in scientific research to embrace further challenges. My project would have been much harder to complete without the help from all the incredible past and present members of Markowitz lab. A special thank goes to Dr. Steve Fink and Dr Kishore Guda for all their support on my project and patience in teaching me countless techniques and ways of scientific thinking; Dr. Jerome F Sah, Dr Min Yan, Dr Baozhong Xin and Dr Hui Xiao for good-spirited discussions and excellent advices in presentation; James Lutterbaugh for preparing