EFFECTS OF RADIATION EXPOSURE ON LUNG CARCINOGENESIS APPROVED BY SUPERVISORY COMMITTEE ______________________________ Jerry W. Shay, Ph.D. Professor of Cell Biology ______________________________ David A. Boothman, Ph.D. Professor Simmons Cancer Center ______________________________ John D. Minna, M.D. Professor of Internal Medicine ______________________________ Michael D. Story, Ph.D. Associate professor of Radiation Oncology DEDICATION Dedicated to my lovely wife Nikki for all of her love and support. EFFECTS OF RADIATION EXPOSURE ON LUNG CARCINOGENESIS by OLIVER DELGADO DISSERTATION Presented to the Faculty of the Graduate School of Biomedical Sciences The University of Texas Southwestern Medical Center at Dallas In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY The University of Texas Southwestern Medical Center at Dallas Dallas, Texas December, 2009 Copyright by OLIVER DELGADO, 2009 All Rights Reserved ACKNOWLEDGEMENTS I would like to acknowledge and thank first and foremost my mentors, Jerry Shay and Woody Wright. The complementarity of your distinctive thoughts and approaches provided me with an unparalleled environment to learn and mature as a young scientist. I would like to thank my thesis committee: Dr. David Boothman, Dr. John Minna, and Dr. Michael Story for their comments and support throughout the years. In addition, thank you to Dr. James Richardson who taught me to see the order within an H&E section. Thanks to all the current and former Shay/Wright lab members that have made my time in the lab both enjoyable and challenging. In particular, I would like to thank David Minna, Suzie Hight, Kimberly Batten, Erin Kitten, Phil Smiraldo, Andres Roig, Richard MacDonnell, Gail Marian, and Aadil Kaisani for their help and/or advice with one or all of my projects. Thank you to the entire UTSW NSCOR team; especially to those that ventured with me to the deep reaches of space (I mean Brookhaven National Laboratory). I would like to give a special thank you to my wife, Nikki. Without your love, patience, and support throughout these years I would have been lost. Last but definitely not least, I would like to acknowledge and thank my parents, Armando and Minervina Delgado. Everything I am and have achieved is due to you. v EFFECTS OF RADIATION EXPOSURE ON LUNG CARCINOGENESIS OLIVER DELGADO, Ph.D. The University of Texas Southwestern Medical Center at Dallas, 2009 JERRY W. SHAY, Ph.D. Lung cancer is one of the most prevalent forms of cancer in both men and women with over 1.3 million annual related deaths worldwide. Analysis of several human populations exposed to radiation reveals that the lung is remarkably susceptible to the carcinogenic effects of radiation exposure. The considerable lung surface area and slow rate of epithelial turnover may have causal roles in this vulnerability. This may be due to the increased probability vi vii that a progenitor cell of the lung, which is proposed to be the cancer-initiating cell, may acquire multiple carcinogenic alterations from radiation exposure. Currently, the lung is believed to have several facultative progenitor cells, situated throughout the lung epithelium, that are regionally restricted in their regenerative capacity. Normal human bronchial epithelial cells (HBECs), immortalized through the expression of Cdk4 and hTERT, provide a sustainable cell reagent for the evaluation of the radiation effects in vitro. These HBECs retain a novel multipotent capacity in vitro (capable of differentiating into both central and peripheral lung cell types) and thus may represent an unrestricted progenitor of the adult lung that resembles an embryonic progenitor. Studies to determine whether the differentiation state influences radiation exposure effects, such as DNA damage and repair, are ongoing. As cellular responses change upon the acquisition of oncogenic mutations, the effects of fractionated or acute radiation exposure on lung carcinogenesis in vivo were determined utilizing the transgenic LA1 K-ras mouse model of lung cancer compared to wildtype littermates. Radiation-induced carcinogenesis is a major concern not only for cancer patients being treated with therapeutic radiation but also for astronauts on long-term space missions. X-ray radiation did not affect the incidence or progression of lung carcinogenesis in this mouse model of lung cancer. High-energy 56Fe- particle irradiation (a type of radiation present in deep space), however, significantly increased the incidence of invasive carcinoma when administered as a viii fractionated dose but not as a single acute dose. These results demonstrate that pre-initiated lesions may be more susceptible to malignant transformation upon exposure to radiation. Thus, radiation may have an impact on both lung cancer initiation and progression. TABLE OF CONTENTS TITLE ................................................................................................................... i DEDICATION ..................................................................................................... ii TITLE PAGE ...................................................................................................... iii COPYRIGHT ...................................................................................................... iv ACKNOWLEDGEMENTS ..................................................................................v ABSTRACT ........................................................................................................ vi TABLE OF CONTENTS .................................................................................... ix PRIOR PUBLICATIONS ................................................................................. xiii LIST OF FIGURES .......................................................................................... xiv LIST OF TABLES ........................................................................................... xvii LIST OF APPENDICES ................................................................................. xviii LIST OF ABBREVIATIONS ........................................................................... xix CHAPTER ONE: INTRODUCTION ...................................................................1 1.1. Basic Lung Biology ...............................................................................1 1.2. Lung Development...............................................................................10 1.3. Stem Cells of the Lung: Homeostasis and Repair ...............................16 1.4. Lung Cancer, Mouse Models of Lung Cancer, and Lung Cancer Stem Cells .....................................................................................................35 1.5. Radiation and Lung Cancer .................................................................46 ix CHAPTER TWO: MULTIPOTENCY OF IMMORTALIZED HUMAN BRONCHIAL EPITHELIAL CELLS ................................................................53 2.1. Introduction ..................................................................................................53 2.2. Results ..................................................................................................59 2.2.1. In Vitro Analysis of HBEC3 KT Potency ...................................59 2.2.2. Transcriptional Analysis of HBEC3 KT Cell Line ....................64 2.2.3. Development of a 3D Differentiation Model Using the HBEC3 KT Cell Line ....................................................................................68 2.3. Discussion ............................................................................................72 2.4. Materials and Methods .........................................................................79 CHAPTER THREE: CHARACTERIZATION OF IN VITRO TRANSFORMED HBEC3 KT POPULATION AND CLONES ISOLATED IN SERUM AND SERUM-FREE ....................................................................................................84 3.1. Introduction ..........................................................................................84 3.2. Results ..................................................................................................89 3.2.1. Analysis of 3D Differentiation Capacity of in vitro Transformed HBEC3 KT Cells .......................................................................89 3.2.2. Characterization of in vitro Transformed HBEC3 KT Clones Isolated in Presence or Absence of Serum ................................90 x xi 3.2.3. Evaluation of Tumorigenicity of in vitro Transformed HBEC3 KT Clones Isolated in Serum ...........................................................97 3.3. Discussion ............................................................................................98 3.4. Materials and Methods .......................................................................102 CHAPTER FOUR: INVASIVE CANCER INDUCED BY RADIATION IN LA1 K-RAS MOUSE MODEL OF LUNG CANCER .............................................106 4.1. Introduction ........................................................................................106 4.2. Results ................................................................................................111 4.2.1. Effect of Acute or Fractionated Doses of 1.0 Gy X-Rays of 56Fe- Particles on Wildtype and LA1 KRAS Mice ............................111 4.2.2. Genomic Analysis of Pulmonary Radiation Response to Irradiation ...............................................................................120 4.2.3. Determination of Relative