THE CCAAT-BOX BINDING TRANSCRIPTION FACTOR, NUCLEAR FACTOR-Y (NF-Y) REGULATES TRANSCRIPTION OF HUMAN ALDO-KETO REDUCTASE 1C1 (AKR1C1) GENE A Dissertation Submitted to the Temple University Graduate Board In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy By Rajash Pallai May 2010 Examining Committee Members: Hemant K. Parekh, Ph.D, Advisory Chair, Department of Pathology and Laboratory Medicine Barrie Ashby, Ph,D, Department of Pharmacology Fayez F. Safadi, Ph.D, Department of Anatomy and Cell Biology Raghava Potula, MHA, Ph.D, Department of Pathology and Laboratory Medicine Iraimoudi S. Ayene, Ph.D, The Lankenau Institute for Medical Research © Copyright 2010 by Rajash Pallai ii ABSTRACT The CCAAT-box binding transcription factor, Nuclear Factor-Y (NF-Y) regulates transcription of human aldo-keto reductase 1C1 (AKR1C1) gene Rajash Pallai Doctor of Philosophy Temple University, 2010 Doctoral Advisory Committee Chair: Hemant K. Parekh, Ph.D. Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Whilst, several phase II drugs as well as endogenous & exogenous steroids/steroid metabolites have been identified as inducers of gene transcription, the cellular transcription factors controlling the expression of AKR1C1 are incompletely elucidated. Herein, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene that controls its transcription. The 5’ flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5’-flanking, ~3.0 kb region of the human AKR1C1 gene identified the region between -128 to -88 as the minimal proximal promoter essential for basal transcription of AKR1C1 in human ovarian (2008 & 2008/C13*), lung (H23 & A549) and liver carcinoma (HepG2) cells. Antioxidant response elements (ARE) have been shown to modulate the transcription ofv genes coding for phase II drug metabolizing enzymes. Cloning of the ARE upstream of iii the AKR1C1 proximal promoter resulted in increased transcription in human lung adenocarcinoma and liver hepatoblastoma cells but not in human ovarian carcinoma cells. Further, ARE increased the induction of the AKR1C1 gene in response to treatment with phase II drug inducers. However, ARE did not induce the transcription of AKR1C1 gene promoter in the presence of cisplatin in any of the cell lines. A computational analysis utilizing the Alibaba 2.0 on the proximal AKR1C1 gene promoter region was performed to identify potential transcription factor binding sites. Based on this analysis, a set of potential, putative transcription factor binding sites for Oct1, Sp1, Cp-1/NF-Y, CEBP, p40X, USF, NF1 and AP-2 were identified in the region -180 to -88 of the AKR1C1 gene promoter. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were involved in controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at -109ATTGG-105 of the AKR1C1 gene. Chromatin immunoprecipitation (ChIP) analysis confirmed the in vivo association between NF-Y and human AKR1C1 gene promoter in human ovarian, lung and liver carcinoma cells. Further, ectopic expression of NF-Y’s increased the AKR1C1 gene transcription, whereas expression of a dominant-negative NF-YA or knockdown of NF-YA by siRNA transfection, decreased the AKR1C1 gene transcription. A 2-fold increase in AKR1C1 transcription was observed specifically in cisplatin-treated 2008 cells that was CCAAT box-dependent. These results indicate that NF-Y regulates basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells and cisplatin-induced transcription in human ovarian carcinoma cells. iv DEDICATION “Dedication is not what others expect from you, it is what you can give to others” This dissertation is gratefully dedicated to my family, especially…. To my parents Madhava Rao Pallai and Hemalatha Pallai. According to a Sanskrit adage “Matha, Pitha, Guru, Deivam” which means “Mother, Father, Teacher, God. Mother is the one who brings us to this world and father is the one who guides us to the teacher and it’s the teacher who later points us to God, which can be our “ real self”. It wouldn’t be possible to accomplish my present work without the help, encouragement, confidence, support and unconditional love from my parents. To my younger brother Suredraa Pallai. You have been instrumental in giving me the courage and support in taking care of our parents. My better half, Preetha Bheema Rao. You have provided me an unconditional love and support which has really increased my confidence and changed me to look forward for the best of the things that life has to offer. The last couple of years its been you, who has been the driving force for me to successfully endure the test of life. v ACKNOWLEGEMENTS This dissertation is the result of collective efforts of a number of people who have assisted and supported directly or indirectly during this doctoral study. To these people, I owe my gratitude and thanks. First of all, I would like to sincerely thank my thesis advisor Dr. Hemant K. Parekh for his guidance and the support throughout the project. It has been a tremendrous journey for me to walk down the path of research as a graduate student in the Deparment of Pathology. Right from the day one when I meet Dr. Parekh in the cross roads of research looking for a direction to attain my goal he has really passed on his experience and philosphoy of research with a calm and composed attitude. One thing which makes me work more and more inspite of ups and downs in research is his saying “All that one needs is few good days in an year to be really successful in research”. This has made me to look forward for those few good days to accomplish even the hardest tasks. As a mentor, Dr. Parekh had enthrused me with great confidence and freedom, which has shaped me up to work independently and develope problem solving skills with an broad outlook. Once again I would really like to thank him for guiding me in the right direction to be successful in accomplishing my professional goal. I would also like to thank my thesis advisory committee: Dr. Barrie Ashby for his consistent assistance in helping me to come over the hinderences faced during the intial phase of graduate studies. I really need to mention that without his support it wouldn’t have been possible for me to survive and thrive as a successful graduate student. I also want to thank Dr. Fayez Safadi and Dr. Raghava Potula for their support, guidance and vi time over the years. I would also like to thank my external examiner, Dr. Iraimoudi S. Ayene for accepting and taking the time to be a part of my thesis committee. I also like to extend my thanks to my collaborator Dr. Henry Simpkins, who has been generous in providing financial assistantship during the initial phase of the project. I also want to thank Dr. Satya P. Kunapuli and Dr. Koneti Rao for granting access to the charge-coupled device camera & luminometer instrumentation. My heartfelt special thanks to Sharon L. Perry, for helping me in ordering of chemicals and reagents, I think without her help not a single day I would have been able to work in the laboratory. I would also like to thank Sandra M. Harper for her help with graduate studies and Sandra would be happy that I wouldn’t be bothering her with course registration and tution waviers!!! I need to mention Gideon Owate a wonderful person to interact with. He has been a good friend. I would sugget anyone who wants an opinion on cars to approach Gideon as he has a vast knowledge. Gideon has been a really nice friend and has shared his wonderful experiences in life. I would like to thank Mahesha Adikari and Dr. Jianli Chen, who have shared their work space with me. vii TABLE OF CONTENTS Page ABSTRACT .................................................................................................................... iii DEDICATION.....................................................................................................................v ACKNOWLEGEMENTS.................................................................................................. vi LIST OF TABLES............................................................................................................ xii LIST OF FIGURES ......................................................................................................... xiii LIST OF ABBREVIATIONS............................................................................................xv CHAPTER 1. INTRODUCTION ...............................................................................................1 1.1 Aldo-keto reductases (AKRs)............................................................................1 1.2 Structural features of AKRs...............................................................................3 1.3 Chromosomal localization, distribution and function of human AKRS............3 1.3.1 Aldehyde reductase (AKR1A1).......................................................3 1.3.2 Aldose reductase (AKR1B1) ...........................................................4 1.3.3 Delta-4-3-ketosteroid-5-beta reductase (AKR1D1).........................5 1.3.4 Kv Proteins (AKR6)……………………………………………..5 viii 1.3.5 Aflatoxin