INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter lace; while others may be from any type o f computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back o f the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell A Howell Information Company 300 North Zeeb Rood, Ann Alter MI 48106-1346 USA 313/761-4700 800/321-0600 NEUROBLASTOMA, A DEFECT IN NEURAL CREST DEVELOPMENT: SOMATOSTATIN RECEPTOR EXPRESSION AND CHARACTERIZATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anne Robinson Albers, B. A. ***** The Ohio State University 1996 Dissertation Committee Approved by Georgia A. Bishop, Ph.D. * A Philip R. Johnson, M.D. sJuJJL- QIJ ja/c y A* \ jlJUJ L X i P M. Sue O’Dorisio, M.D., Ph.D. ( Advisor Alan J. Yates, M.D., Ph.D. Neuroscience Graduate Program UMI Number: 9639178 UMI Microform 9639178 Copyright 1996, by UMI Company. All rights reserved. This microform edition b protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT The hypothesis that the neuropeptide somatostatin and somatostatin receptors play a role in the biology of neural crest development was tested by studying neuroblastoma. A second hypothesis, that studies of somatostatin receptors in neuroblastoma may lead to therapeutic advances in clinical neuroblastoma, was also tested. In vitro gene analysis, in vitro pharmacologic characterization, and in vivo growth studies, pharmacologic characterization, and analysis of gene expression were pursued. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) was used to analyze the expression of somatostatin and all five somatostatin receptor subtypes (ssti - sst5) in 34 tumor tissue samples from 26 patients with neuroblastoma. All 34 tumor specimens expressed a control gene, c-abl, and ssti ; 33/34 samples expressed SS; sst 2 was expressed in 29/34 samples, and sst 3 , ssU, and sstj variably expressed. Pharmacologic analysis of binding affinities of somatostatin 14 (SS 14) and somatostatin congeners octreotide, CH275, and WOC3b, was studied in SKNSH neuroblastoma cell lines stably transfected with ssti and sst2. SSu bound with high affinity to the SKNSH/ssti and SKNSH/sst 2 neuroblastoma cell lines. Octreotide bound with high affinity only to SKNSH/sst 2 . CH275 bound with high affinity only to SKNSH/ssti- WOC3b bound with high affinity to both SKNSH/ssti and SKNSH/sst 2 . No significant difference in tumorigenesis was observed between the cell lines SKNSH, SKNSH/ssti, and SKNSH/sst 2 when used for a xenograft model of neuroblastoma. In vivo binding characteristics of the SKNSH, SKNSH/ssti, and SKNSH/sst2 cell lines demonstrated variable binding by receptor specific analogues. In vivo gene expression of upregulated sst was preserved. These studies present the first molecular analysis of the expression of the five sst in neuroblastoma; and demonstrate that neurpeptide-receptor interaction plays a role in neuroblastoma, and by inference, in neural crest development. Additionally, the application of receptor biology to detection of and/or therapy for neuroblastoma offers exciting prospects for improving the clinical management of neuroblastoma. Dedicated to M. Sue O’Dorisio ACKNOWLEDGMENTS I would like to thank my family for their encouragement, support, and inspiration. This dissertation is dedicated to MSO, whose confidence in me and enthusiasm for her work and life are the foundation for this work and inspiration for my career. I am grateful to Dr. Debbie Martinez, Monica Summers, Dr. Gail Wenger, and Dr. Anne Lewis for sharing techniques, styles, and ideas with me. I am grateful for the efforts of Dr. Thomas M. O’Dorisio and Dawn Wray who helped me become a poised, engaging, public speaker. I would like to acknowledge Dr. Steven Qualman for providing not only his own time and ideas but also for making available the resources of the Cooperative Human Tissue Network and the help of the technicians in the department of anatomic pathology. I also wish to thank Julia M. Kim, Steve Kirkby, Chris Elliott, Jeff Sail, Leigh Sotos, Marsha Hauger, Dr. Sang Park, and Dr. Michael Fruehwald who have helped me immeasurably with this work. I would finally tike to acknowledge Pat Davis, Sharon Palko, and Brad Baker for their help in preparing my dissertation's final form. This work was supported by the Howard Hughes Medical Institute, by the Neuroscience Graduate Program at The Ohio State University, and by NCI ROl Grant # CAM 177 to MSO. VITAE July 12, 1968 ........................................................................................ Born - Columbus, Ohio 199 0 ................................................................................. B.A. English, Yale University, New Haven, Connecticut 1991 - present ........................................................................Medical student, The Ohio State University College of Medicine Graduate Student, The Ohio State University Neuroscience Graduate Program PUBLICATIONS 1. A.R. Albers, M.S. O’Dorisio, "Clinical Use of Somatostatin Analogues in Paediatric Oncology.” Digestion., 1996; 57(suppl 1):38-41. 2. A.R. Albers, M.S. O’Dorisio, and A.J. Yates, “Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of somatostatin receptor expression (SST) in neuroblastoma, Soc. Neurosci. Abst., 21 (1995) 2133 (Abstract). FIELDS OF STUDY Major field: Neuroscience TABLE OF CONTENTS Page Dedication ................................................................................................................................... iv Acknowledgments ................................................................................. v V itae............................................................................................................................................... vi List of Tables ..............................................................................................................................viii List of Figures ...............................................................................................................................ix Chapters: 1. Introduction ...................................................................................................................... 1 2. Background and Significance ........................................................................................ 3 3. Materials and Methods ................................................................................................. 42 4. R esults................................................................................... 72 5. Discussion ................................................................................................ 113 References .................................................................................................................................. 122 LIST OF TABLES Table Page 2.1 Nomenclature for human somatostatin receptor subtypes (ssti - sst;) .................... 23 2.2 Expression of sst] through sst; in mouseM and ratR b rain ..........................................24 2.3 Expression of ssti through sst; in rat peripheral tissues .............................................27 4.1 Patient prognostic factors and outcome ....................................................................... 78 4.2 Primer design for the c-abl, human p actin (HPA), somatostatin (SS) and somatostatin receptor genes 1-5 (ssti.j) .............................................................. 80 4.3 Probe sequences for Southern confirmation of RT-PCR analysis ...........................81 4.4 Expression of ssti - sst5, somatostatin (SS), and c-abl in control tissues and neuroblastoma cell lines ..........................................................................81 4.5 COS-7 expression of pcDNA3/ssti, pcDM 8 /sst2 . and pcDNA 3 /sst2 , evaluated by total binding to [l 2 5 I-tyrn ]-somatostatini 4 ...........................................87 4.6 Affinity (K d ) of peptides for ssti and sst 2 .......................................................... 97 viii LIST OF FIGURES Figure Page 4.1 Total RNA isolation ........................................................................... 72 4.2 RT-PCR analysis of sst 2 and P actin expression in IMR32 and SKNSH total RNA ..................................................................................................74