HUMAN CARBOXYLESTERASE 2 SPLICE VARIANTS: EXPRESSION, ACTIVITY, AND ROLE IN THE METABOLISM OF IRINOTECAN AND CAPECITABINE Marissa Ann Schiel Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Biochemistry and Molecular Biology, Indiana University February 2009 Accepted by the Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. William F. Bosron, Ph.D., Chair E. Gabriela Chiorean, M.D. Doctoral Committee David A. Flockhart, M.D., Ph.D. Maureen A. Harrington, Ph.D. August 28, 2008 Sonal P. Sanghani, Ph.D. ii To my Poppa, Who encouraged me to finish and do my best. iii ACKNOWLEDGEMENTS I am sincerely thankful for all the help and encouragement I have received while pursuing my graduate education. I would like to gratefully acknowledge the following individuals: Dr. William Bosron for his passion for both science and education. I met Dr. Bosron on my very first visit to IUSM, and I was beyond pleased when I found a place in his lab. His wisdom and generosity truly enhanced my graduate experience. Dr. Sonal Sanghani for her knowledge and guidance during every day of this journey. I am grateful that she is both my mentor and my friend. Dr. Maureen Harrington, committee member and co-director of the MSTP. Her mentorship has been invaluable as I pursued both my graduate and medical studies. With her guidance, I happily did a rotation and found a home in the Bosron lab. She and Dr. Sanghani are my role models for being a strong female scientist. Dr. David Flockhart for the wisdom and thoughtfulness he brought to each committee meeting. His questions were encouraging and thought-provoking, and they always led to a step forward in my research. Dr. Gabi Chiorean, my translation research mentor, for her kindness and enthusiasm during our collaboration on the HOG GI03-53 project. I admire her passion for medicine and clinical research. Dr. Paresh Sanghani for his support in the lab, for sharing his knowledge of protein biochemistry, and for completing the circular dichroism studies. iv Wilhelmina Davis, my lab mate, for her assistance with “all things protein,” especially protein purification and westerns. I am truly thankful for her encouragement and friendship. Sharry Fears, my lab mate, lunch buddy, and fellow Big Ten supporter, for her work on the sub-cellular localization studies. I am grateful for all of her help, support, and friendship. Scheri-lyn Green for all of her work on PCR and cloning. I look forward to working with her in the future as a physician colleague. Lan Min Zhai for her assistance with cloning, protein purification, and cell culture and for her ability to always make me smile. Susan Perkins from the Indiana University Cancer Center for performing kurtosis analysis on the tissue sample data. All of the friends I made on third floor of the BRTC including Darlene Lambert, Jack Arthur, Bradley Poteat, Alice Nakatsuka, Oun Kiev, Amy Dietrich, Pam Kelley, and the members of the Goebl and Harris labs. I appreciate the knowledge, advice, humor and commiserating we have all shared. Dr. Wade Clapp, Jan Receveur, and my fellow combined degree students for their friendship, support and advice during this seven year journey. Dr. Mike Zimmer and Dr. Hendrick Szurmant whose enthusiasm for science while graduate students at the University of Illinois inspired me to pursue a graduate degree in research. v My extended family, otherwise known as my entourage, my grandparents June Collins and Zvonimir and Maria Jugovic; my aunts and uncles Bob and Linda Reiff and John and Cheryl Jugovic; and my cousins Erin Dunivan and Kristin and Scott Petherick. Your love and support throughout my life and education has been and continues to be incredible. My brother Robbie Collins for challenging me and supporting me in ways only a sibling could. I am grateful that you are both my brother and my friend. My parents Bob and Mary Ann Collins for always loving me, supporting me and inspiring me to do my best. I am truly blessed to have such remarkable parents. My husband Zack Schiel for sharing with me in both the joys and frustrations of this adventure. I am genuinely grateful for his boundless love and support without which I would not have happily made it this far. vi ABSTRACT Marissa Ann Schiel Human Carboxylesterase 2 Splice Variants: Expression, Activity, and Role in the Metabolism of Irinotecan and Capecitabine Carboxylesterases (CES) are enzymes that metabolize a wide variety of compounds including esters, thioesters, carbamates, and amides. In humans there are three known carboxylesterase genes CES1, CES2, and CES3. Irinotecan (CPT-11) and capecitabine are important chemotherapeutic prodrugs that are used for the treatment of colorectal cancer. Of the three CES isoenzymes, CES2 has the highest catalytic efficiency for irinotecan activation. There is large inter-individual variation in response to treatment with irinotecan. Life-threatening late-onset diarrhea has been reported in approximately 13% of patients receiving irinotecan. Several studies have reported single nucleotide polymorphisms (SNPs) for the CES2 gene. However, there has been no consensus on the effect of different CES2 SNPs and their relationship to CES2 RNA expression or irinotecan hydrolase activity. Three CES2 mRNA transcripts of approximately 2kb,3kb, and 4kb have been identified by multi-tissue northern analysis. The expressed sequence tag (EST) database indicates that CES2 undergoes several splicing events that could Δ generate up to six potential proteins. Four of the proteins CES2, CES2 458-473, CES2+64, Δ CES2 1-93 were studied to characterize their expression and activity. Multi-tissue northern analysis revealed that CES2+64 corresponds to the 4kb and 3kb transcripts while Δ Δ CES2 1-93 is located only in the 4 kb transcript. CES2 458-473 is an inactive splice variant vii that accounts for approximately 6% of the CES2 transcripts in normal and tumor colon tissue. There is large inter-individual variation in CES2 expression in both tumor and normal colon samples. Characterization of CES2+64 identified the protein as normal CES2 indicating that the signal peptide is recognized in spite of the additional 64 amino acids at the N-terminus. Sub-cellular localization studies revealed that CES2 and Δ CES2+64 localize to the ER, and CES2 1-93 localizes to the cytoplasm. To date CES2 SNP data has not provided any explanation for the high inter-individual variability in response to irinotecan treatment. Multi-tissue northern blots indicate that CES2 is expressed in a tissue specific manner. We have identified the CES2 variants which correspond to each mRNA transcript. This information will be critical to defining the role of CES2 variants in the different tissues. William F. Bosron, Ph.D. viii TABLE OF CONTENTS List of Tables .............................................................................................................xi List of Figures ............................................................................................................xii List of Abbreviations .................................................................................................xiv INTRODUCTION I. Carboxylesterase genes and enzyme functions ...........................................2 II. CES2 structure and polymorphisms............................................................6 III. Gene splicing ..............................................................................................10 IV. Colorectal cancer ........................................................................................12 V. Irinotecan ....................................................................................................14 VI. Capecitabine ................................................................................................18 VII. Research objectives.....................................................................................21 METHODS I. Materials .....................................................................................................22 II. Tissue-specific expression of CES2 splice variants ....................................23 III. Analysis of CES2 and CES2∆458-473 in paired tumor and normal colon samples ..............................................................................................25 IV. Characterization of the CES2Δ458-473 variant ...............................................29 V. Characterization of the CES2+64 variant .....................................................31 VI. Sub-cellular localization of CES2 variants .................................................38 VII. The role of CES2, CES1, TOPO I, TP, TS, DPD, β-GUS, and UGT1A1 in the inter-individual variation in response to treatment of rectal cancer with irinotecan and capecitabine .................................................................41 ix RESULTS I. Tissue-specific expression of CES2 splice variants ....................................47 II. Analysis of CES2 and CES2∆458-473 in paired tumor and normal colon samples ..............................................................................................48 III. Characterization of the CES2Δ458-473 variant ...............................................58 IV. Characterization of the CES2+64 variant .....................................................63 V. Sub-cellular localization of CES2 variants .................................................76