From chromaffin cells to Phaeochromocytoma: insight into the sympathoadrenal cell lineage

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1 From chromaffin cells to Phaeochromocytoma: insight into the sympathoadrenal cell lineage Susannah Cleary This thesis was submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (Biomedical Science), Murdoch University. July 2007 i

2 Declaration I declare that this thesis is my own account of my research and contains work that has not been previously submitted for a degree at any other tertiary educational institution. Susannah Cleary ii

3 Abstract Chromaffin cells are a modified post-ganglionic sympathetic neuron, which synthesise and secrete catecholamines. The neoplastic transformation of chromaffin cells is demonstrated by the tumour phaeochromocytoma, a functional tumour that recapitulates the normal role of chromaffin cells by synthesising, storing and releasing excess catecholamines. Within this thesis we have explored several aspects of chromaffin cell and phaeochromocytoma tumour biology, including the specific expression of key sympathoadrenal markers such as the noradrenaline transporter (NAT), neuropeptide Y (NPY) and chromogranin A (CGA) in normal human and mouse chromaffin cells versus phaeochromocytomas of human and mouse origin. Catecholamine-mediated signalling in chromaffin cells is terminated by the sequestration of extracellular catecholamines back into the cell via the noradrenaline transporter (NAT). Following observations that within the rat adrenal medulla, NAT is expressed in PNMT-positive chromaffin cells we explored whether this pattern of expression is also present in the human adrenal medulla. While we successfully established that NAT and PNMT are co-localised, we also found that all human adrenal chromaffin cells are PNMT-positive. In the rat, NAT is also observed within the cytoplasm and has been suggested to be associated with secretory vesicles, thus using the secretory vesicle marker, CGA, we demonstrate that NAT is associated with secretory vesicles. However, in contrast to our findings within the normal chromaffin cells, in situ NAT expression in human phaeochromocytoma tumour samples was distorted, with observed changes including the level and type of staining observed, and disruptions to the strict NAT-CGA association observed in the normal adrenal. Continuing our theme of NAT, we investigated if pre-treating the phaeochromocytoma PC12 cell line with the chemotherapy drug cisplatin had an effect on the expression of NAT, to give an indication of the efficacy of this compound in the iii

4 treatment of metastatic phaeochromocytoma with radiolabelled 131 Iodometabenzylguanidine ( 131 I-MIBG), a noradrenaline analogue which can be incorporated into phaeochromocytoma tumour cells though uptake through NAT. The premise of this study is derived from previous work in which neuroblastoma cells pre-treated with cisplatin were more responsive to ( 131 I-MIBG) accumulation due to increased activity and expression of the transporter. Thus we treated PC12 cells for 24-hours in a range of cisplatin concentrations and measured the effect on NAT expression. However, unlike the findings in neuroblastoma cells, in our study, we did not observe an effect of cisplatin pretreatment on NAT activity or expression in PC12 cells. Upto 30% of phaeochromocytoma arise as apart of a hereditary syndrome. The von Hippel-Lindau (VHL) syndrome, due to germline mutations to the VHL gene, and Multiple Endocrine Neoplasia type 2 (MEN 2), due to germline mutations to the RET gene represent two examples of hereditable endocrine disorders where phaeochromocytoma is a presenting feature. Notable differences in clinical presentation and tumour biology have been identified in phaeochromocytomas from patients with VHL and MEN 2. These differences prompted us to explore whether these observations extend to the chromaffin granule constituents, NPY and CGA. Patients with MEN 2 disease have a greater incidence of hypertension than patients with VHL disease, MEN 2 are characterised by an adrenergic phenotype (produce predominantly-adrenaline), whereas VHL phaeochromocytomas are noradrenergic (produce predominantly-noradrenaline). Neuropeptide Y, which has powerful vasoactive properties capable of significantly elevating blood pressure, is stored and released with catecholamines and is thought to be associated with PNMT-positive chromaffin cells. Thus, we questioned whether the differences in the symptomatology between VHL and MEN 2 patients may be related to differences in NPY expression between the two groups, and whether any differences in NPY relate to adrenaline and/or PNMT content, or are linked to hereditary factors. Thus we compared tumour samples from four cohorts of patients: (i) adrenergic versus noradrenergic phenotype, (ii) hereditary versus no hereditary syndrome. Results demonstrated that although tumour NPY levels (mrna and peptide) correlate with PNMT expression and/or adrenaline content, when NPY expression was iv

5 compared between groups of patients (adrenergic vs noradrenergic; hereditary versus nonhereditary) difference in NPY levels were only significant between VHL and MEN 2 tumour and not between sporadic adrenergic and noradrenergic Immunohistochemistry also supported the above observations. Hence, we concluded that NPY expression in all groups of phaeochromocytoma examined in this study, this effect is not related to tumour biochemical phenotype but rather appears to be a specific unique trait of VHL phaeochromocytomas. Continuing our theme of the possible differential expression of chromaffin granule constituents between VHL and MEN 2 patients, we also investigated CGA levels in plasma and tumour samples. Given, VHL tumours possess less chromaffin granules than MEN 2 tumours, and CGA has been implicated as a key director of secretory vesicle biogenesis we investigated whether CGA was differentially expressed between VHL and MEN 2 tumours. We found CGA expression was significantly greater in MEN 2 tumours (mrna; 3-fold, and protein; 20-fold), with western blot confirming this trend. We also found that plasma CGA was greater in MEN 2 patients but not significantly, consequently, we explored the co-variables tumour size and tumour secretory activity (measured by plasma catecholamine concentrations), which influence plasma CGA and found that tumour size and plasma CGA are related but there was no influence of genotype on this relationship. In contrast, plasma CGA was significantly related to tumour secretory activity and the effect of genotype on this relationship narrowly missed significance, but when we expressed plasma CGA as a ratio of plasma catecholamines, plasma CGA was 2-fold greater in MEN 2 patients than VHL patients. Thus despite the tendency of phaeochromocytomas from VHL disease to readily and continuously release their catecholamine stores, plasma CGA levels still appeared to be higher in MEN 2 patients. Finally, we examined whether the expression of NPY, Leu-enkephalin (Leu-Enk), NAT and the vesicular monoamine transporters type 1 and 2 (VMAT1 and VMAT2,), in normal mouse adrenal glands, and in histologically-confirmed adrenal phaeochromocytomas generated by injected nude mice with a phaeochromocytoma (MPC) cells line. The results of this study established that similar to the rat and human NAT expression is preferentially localised with PNMT within mouse chromaffin cells, while v

6 VMAT1 and NPY are found in both PNMT-negative and PNMT-positive cell populations, although expression of NPY was reduced in PNMT-negative cells. In contrast, both VMAT2 and Leu-Enk were found in PNMT-negative noradrenergic cells, and VMAT2 was present in all noradrenergic chromaffin cells while Leu-Enk was observed in a subpopulation of noradrenergic chromaffin cells. In contrast to the normal adrenal but similar to our findings in human phaeochromocytoma, the pattern of marker expression within adrenal phaeochromocytoma lesions of MPC-injected mice are severely disrupted related to both the level of expression of the respective markers, and association with PNMT within the tissue. Thus, the experimentally generated phaeochromocytoma mouse model provides a valuable tool in studying human phaeochromocytoma. The data presented in this thesis further validate the heterogeneity observed in many aspects of phaeochromocytoma tumour biology, including the expression several chromaffin cell markers such as NAT, NPY and CGA. The altered expression of these markers may contribute to the clinical picture of these tumours, particularly relating to hereditary phaeochromocytoma from VHL and MEN 2 disease. vi

7 Acknowledgements To my two supervisors, Jackie and Graeme, I owe so much. Thank you to both of you for putting up with my (sometimes) tempestuous behaviour. This never would have possible without the two of you. While I don t think that there could be more different styles from each of you, working under the both of you has been a rewarding and fabulous experience. Jackie, our time together has been great, and I ll miss being in your group. Graeme, sharing an office with you was great fun even if you are Dr. Critical, all the criticisms came from the right place for the right purpose. Thank you to both of you for giving me to opportunity to work at NIH it changed my life. Muito obrigada. I want to express my gratitude to my fellow Molecular Neurobiology Lab members (past and present) for all the help you ve given me along the way, especially to Kelly for everything you ve given to me as a friend and colleague. To everyone from the School of Veterinary & Biomedical Sciences at Murdoch University who helped me along the way, including Graham Wilcox, Phillip Nichols, Phil Clarke, John Pluske, the boys and girls from Virology and Parasitology I owe you guys so much for the support and time and patience you ve given me (and possibly for listening to me sing in hallways, labs or offices). Thank you to Meredith and Ryan for the support; even across long distances the two of you have been a therapy session that s just a phone call away. God Bless. Thank you to Dave, Courtney, Basil, Ella, Oladi and Tereza from the Clinical Neurocardiology section at NIH. Special thank you also to Thanh for your wealth of assistance and to Richard for all those great times singing in the lab and in the darkroom. Thank you to Karel for your support (financial and otherwise) and to everyone from the Reproductive Biology and Medicine Branch including Karen, Danny, Priya, Shiromi and especially to Lucia, Steffi and Edwin for all their assistance and good times. Thank you to David Christie from the University of Auckland for graciously allowing us to use your fabulous NAT antibody. Without it, this project would never have happened. To Jennifer, Peter from the Center for Information Technology and Abdel from the National Human Genome Research Institute working with you guys was great and you all made those horrible meetings tolerable. Thank you so much for helping with the microarray. To the staff, including Dr. Linehan and Robert Worrell, from the Urologic Oncology Branch for their assistance acquiring tumour samples. Thank You, Thank You, thank you to my wonderful family, especially my wonderful parents. Mum, your strength and resolve are an inspiration. Dad, your moments of insight are a pleasure to be around and a reminder of what lies within. To Scott, although your thoughts went unsaid, they were nonetheless felt, and Georgia your support and encouragement were a constant. And thank you to my second family: Paul and Joanne. And ofcourse to the animals of number 51: The Little Grey Assassin and my beloved Mutation and Hairy Legs. This thesis is dedicated to Justin. We ll share the shelf together. vii

8 Table of Contents Declaration Abstract Acknowledgements Table of Contents Publications arising from this thesis Other publications arising from the period of candidature Declaration of contribution to Chapters containing published or submitted work List of Figures List of Tables ii iii vii viii xiv xiv xv xvii xix Abbreviations Part A The adrenal gland, catecholamines and chromaffin cells 1 I. A brief introduction to the autonomic nervous system 1 II. The adrenal gland: Historical vignette 3 The adrenal cortex 3 The adrenal medulla 5 III. Catecholamines 6 Introduction 6 From tyrosine to catecholamines: the catecholamine biosynthetic pathway 6 Catecholamines elicit their effects on physiology by binding to adrenoceptors 9 Alpha adrenoceptors 10 Beta Adrenoceptors 11 Catecholamine metabolism 12 Dopamine metabolism 12 Noradrenaline and adrenaline metabolism 14 Neuronal vs adrenal catecholamine metabolism 15 IV. The Adrenal Medulla 16 The Developmental Lineage of Chromaffin Cells 17 Are glucocorticoids the key to chromaffin cell development? 20 If glucocorticoids don t govern chromaffin cell development, what factors do? 21 Don t give up on glucocorticoids just yet 24 Cortical chromaffin cell interactions: a reciprocal agreement 25 viii xx

9 In-vitro chromaffin cell models 26 V. The Three R s regulating catecholamines: Release, Reuptake & Recycling 27 Introduction 27 Gettin stuff out: Release 28 Constitutive versus regulated secretion 28 Full Fusion exocytosis 29 Kiss-and-run exocytosis 30 Piecemeal degranulation? 31 Gettin stuff back in. Reuptake: Catecholamine Transporters 32 Catecholamine Transporters 32 Molecular characteristics of catecholamine transporter 32 Regulation of transporter systems 34 Monoamine clearance is monoamine specific or is it? 37 Catecholamine transporters also mediate catecholamine inactivation 38 And adrenaline clearance in chromaffin cells? 39 Alternatives methods of catecholamine reuptake 39 Keepin stuff in. Recycling: Vesicular Monoamine Transporters 41 Leaky Stores 42 VI. Chromaffin Granule Cargo Proteins 43 Introduction 43 Neuropeptide Y 44 Neuropeptide Y receptors 45 Neuropeptide Y: is a sympathetic co-transmitter and a vasoactive peptide 47 Chromogranin A 48 CGA, chromaffin cells and hypertension. 50 Part B Phaeochromocytoma: Clinical characteristic & genetics 51 VII. Perspectives on a clinical enigma 51 Introduction 51 Nomenclature defined 53 Diagnosing the Great Mimic 54 Biochemistry 55 Malignant phaeochromocytoma 58 Localisation 59 Anatomical localisation 59 Functional Localisation 59 Treatment 61 Classical concepts 61 Emerging concepts in treatment 62 Other alternatives for treatment of metatstatic PC or PG 63 VIII. Hereditary phaeochromocytoma: 63 Introduction 64 The von Hippel-Lindau syndrome 64 The VHL phenotype 64 VHL genotype 67 VHL and oxygen sensing pathways 67 Neurofibromatosis Type 1 69 The NF1 phenotype 69 The NF1 genotype 69 Multiple Endocrine Neoplasia Type 2 70 The MEN 2 phenotype 70 The MEN 2 genotype 71 ix

10 The phaeochromocytoma paraganglioma syndrome 72 Succinate Dehydrogenase 72 IX. Five genes and one disease: Development along a common pathway of pathogenesis? 74 Developmental culling Linking the five distinct phaeochromocytoma susceptibility mutations to a common pathway 75 X. Differences between VHL and MEN 2 79 XI. Summary, Aims and Objectives 80 Chapter 2 Materials and Methods 83 I. Ethics Approval 83 Human Samples 83 Animals 83 II. Patient and sample details 84 Patients 84 Plasma Procurement 84 Tumour Procurement 85 Procurement of normal human adrenal tissue 85 Plasma and Tissue Catecholamine Measurements 86 Animals 86 I. Extraction of RNA and Reverse Transcription 87 Extraction of RNA and Reverse Transcription: Human Samples 87 Extraction of RNA and Reverse Transcription: Cell Culture 88 II. Quantitative Polymerase Chain Reaction (Q-PCR) 89 TaqMan Q-PCR: Human tissues 89 SYBR Green Q-PCR: Cultured PC12 cells 89 TaqMan vs SYBR Green Q-PCR 89 Q-PCR Data Analysis 90 Protein extraction for ELISA 91 Western Blot 91 III. Immunohistochemistry 92 General immunohistochemistry protocol: Paraffin embedded tissue sections 92 Free-floating tissue sections 95 Cultured PC12 cells 95 IV. Confocal Microscopy 96 V. Statistical Analysis 97 Chapter 3 Expression of NAT & PNMT in the normal human adrenal medulla and phaeochromocytoma 98 I. Abstract 98 II. Introduction 99 x

11 III. Materials and Methods 102 Tissue procurement and patient samples [ 18 F]-Fluorodopamine PET scanning 103 Immunohistochemistry 103 Confocal microscopy 104 IV. Results 104 TH, PNMT and NAT expression in normal human adrenal samples 104 TH, PNMT and NAT expression in sporadic phaeochromocytoma tumour samples 105 Relationship between NAT and CGA 106 NAT expression in hereditary phaeochromocytomas from VHL and MEN Control sections 114 V. Discussion 119 PNMT expression. 119 What is NAT doing in chromaffin cells? 121 Association between NAT and secretory granules 122 Is aberrant NAT expression clinically significant? 125 But don t forget the VMATs 126 Chapter 4 Effects of Cisplatin on the activity and expression of NAT in PC12 cells 129 I. Abstract 129 II. Introduction 130 III. Materials and Methods 133 Cell Culture 133 [ 3 H]-Noradrenaline Uptake Assays 133 [ 3 H]-Noradrenaline uptake experiments statistical analysis 134 Ribonucleic acid isolation and reverse transcription 134 Quantitative real-time reverse transcriptase-polymerase chain reaction 135 Immunofluorescence 136 IV. Results 137 Effect of Cisplatin on NAT mrna levels 137 Effect of Cisplatin on NAT cellular expression 139 Effect of Cisplatin on NAT functional activity 141 V. Discussion I-MIBG and NAT expression 142 Why combine 131 I-MIBG and chemotherapy, and why chose Cisplatin? 143 Different responses due to different cells 145 Chapter 5 NPY expression in phaeochromocytomas: Relative absence in tumours from patients with VHL syndrome 147 I. Abstract 147 II. Introduction 148 III. Materials and Methods 150 Patients and tumour specimens 150 Quantitative Polymerase Chain Reaction (PCR) 151 xi

12 NPY peptide quantification and extraction 151 Immunohistochemistry 152 Statistics 152 IV. Results 154 Tumour Biochemical Phenotypes 154 PNMT mrna levels 156 NPY mrna levels 156 NPY peptide levels 159 NPY staining in normal human adrenal tissues 161 NPY staining in phaeochromocytoma tumour samples 161 V. Discussion 164 Relationship between NPY and PNMT 164 Could NPY contribute to the differences in the clinical presentation observed between VHL and MEN 2 patients? 166 Chapter 6 CGA expression in phaeochromocytomas associated with von Hippel-Lindau syndrome and multiple endocrine neoplasia type I. Abstract 169 II. Introduction 170 III. Materials & Methods 171 Patients 171 Quantitative Polymerase Chain Reaction (PCR) 171 CGA Enzyme-Linked ImmunoSorbent Assay (ELISA) 172 Western Blot 172 Immunohistochemistry 174 Statistics 174 IV. Results 175 CGA Quantitative PCR and ELISA 175 CGA Western Blot 178 CGA Immunohistochemistry 178 CGA plasma concentrations 180 V. Discussion 183 CGA and secretory granules: observations extended 183 No difference in plasma CGA between groups? Take a closer look 184 What causes decreased CGA in the first place? 186 Can these findings be used clinically? 186 Chapter 7 Differential expression of chromaffin cell markers in the mouse adrenal and adrenal lesions of a novel mouse metastatic phaeochromocytoma model 188 I. Abstract 188 II. Introduction 189 III. Materials and Methods 191 xii

13 Animals 191 Cell Culture and generation of tumour lesions 192 Anatomical and functional imaging 193 Immunohistochemistry 193 Confocal Microscopy 194 IV. Results 195 Anatomical and functional imaging 195 MicroCT imaging and MRI scanning 195 F-FDA PET scanning 195 Gross Necropsy of MPC-treated nude mice 196 Histopathology of Adrenal Lesions 199 Expression of sympathoadrenal markers in the adrenal glands of balb/c mice and control nude mice. 199 Expression of sympathoadrenal markers in adrenals of MPC-injected nude mice 206 Expression of peptide markers: NPY and Leu-ENK 206 Expression of monoamine transporters: NAT, VMAT1 & VMAT2 206 V. Discussion 213 Neuropeptides 213 Monoamine Transporters 216 Phaeochromocytoma animal models 218 New information about the MPNM model 219 Chapter 8 General Discussion, Conclusions and Future Perspectives 222 Is there a local role for adrenal catecholamines? 222 Are noradrenergic chromaffin cells more neuronal than adrenergic chromaffin cells? 224 Arrested development 225 Methodological considerations 228 Chapter 9 References 232 Chapter 10 Appendix: Examination of key neural crest developmental genes in VHL & MEN 2 tumours 268 I. Introduction 268 II. Materials & Methods 269 Oligonucleotide Microarrays 269 Quantitative Polymerase Chain Reaction (PCR) 269 III. Results 270 IV. Discussion 272 xiii

14 Publications arising from this thesis Cleary S, Phillips JK, Huynh T-T, Pacak K, Fliedner S, Elkahloun AG, Munson P, Worrell RA, Eisenhofer, G (2007). Chromogranin A expression in phaeochromocytomas associated with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2. Hormone and Metabolic Research. In press. Cleary S, Phillips JK, Huynh T-T, Pacak K, Elkahloun AG, Barb JJ, Worrell RA, Goldstein DS, Eisenhofer G (2007). Neuropeptide Y expression in phaeochromocytomas: Relative absence in tumours from patients with von Hippel-Lindau syndrome. Journal of Endocrinology. 193: Cleary S, Phillips JK (2006). The norepinephrine transporter and pheochromocytoma. Annals of the New York Academy of Sciences. Aug;1073: Huynh TT, Pacak K, Brouwers FM, Abu-Asab MS, Worrell RA, Walther MM, Elkahloun AG, Goldstein DS, Cleary S, Eisenhofer G (2005). Different expression of catecholamine transporters in phaeochromocytomas from patients with von Hippel-Lindau syndrome and multiple endocrine neoplasia type 2. European Journal of Endocrinology. Oct; 153(4): Cleary S, Brouwers FM, Eisenhofer G, Pacak K, Christie DL, Lipski J, McNeil AR, Phillips JK (2005). Expression of the noradrenaline transporter and phenylethanolamine N-methyltransferase in normal human adrenal gland and phaeochromocytoma. Cell and Tissue Research Dec; 322(3): Other publications arising from the period of candidature Ohta S, Lai EW, Morris JC, Bakan DA, Klaunberg B, Cleary S, Powers JF, Tischler AS, Abu-Asab M, Schimel D, Pacak K (2006). MicroCT for high-resolution imaging of ectopic pheochromocytoma tumors in the liver of nude mice. Int J Cancer. Nov 1;119(9): Dixon DN, Loxley RA, Barron A, Cleary S, Phillips JK (2005). Comparative studies of PC12 and mouse pheochromocytoma-derived rodent cell lines as models for the study of neuroendocrine systems. In Vitro Cell Dev Biol Anim. Jul-Aug; 41(7): xiv

15 Declaration of contribution to Chapters containing published or submitted work Chapter 3 The candidate performed all immunohistochemistry experiments and confocal microscopy analysis components of the study. Candidate was a major contributor to the manuscript. Courtney Reddrop performed pre-absorption experiments with GST-NATM2 fusion protein and the 6-[ 18 F]-Fluorodopamine PET scans were interpreted by Dr. Frederieke Brouwers. Chapter 4 The candidate performed all cell culture, [ 3 H]-noradrenaline uptake assays, Q-PCR, immunohistochemistry experiments and analysis components of the study. Candidate was a major contributor to the manuscript. Clare Auckland contributed to the RNA extraction and Reverse Transcription reactions on the cultured cells Chapter 5 The candidate performed all Q-PCR, peptide extraction, ELISA, immunohistochemistry and confocal microscopy analysis components of the study. Candidate was a major contributor to the manuscript. Thanh-Truc Huynh performed the RNA extraction and Reverse Transcription reactions on the human phaeochromocytoma samples, and the PNMT Q-PCR. Edwin Lai and Patti Sullivan performed the HPLC analyses of tumour and plasma catecholamine concentrations. Chapter 6 The candidate performed all Q-PCR, peptide extraction (for ELISA), immunohistochemistry, ELISA and confocal microscopy analysis components of the study. Candidate was a major contributor to the manuscript. Thanh-Truc Huynh performed the RNA extraction and Reverse Transcription reactions on the human phaeochromocytoma samples. Edwin Lai and Patti Sullivan performed the HPLC analyses of tumour and plasma catecholamine concentrations. Steffi Fliedner performed the protein extractions (for Western Blot analysis) and subsequent Western Blots. xv

16 Chapter 7 The candidate performed all immunohistochemistry experiments and confocal microscopy analysis components of the study. Candidate was a major contributor to the manuscript. Lucia Martiniova performed the anatomical and functional imaging studies (and related data analysis) and also performed the cell culture component of this study. xvi

17 List of Figures Figure 1.1 Glandulae Renibus Incumbentes : The kidney and adrenal gland 4 Figure 1.2 Catecholamine Biosynthetic Pathway 8 Figure 1.3 Pathways of catecholamine metabolism 13 Figure 1.4 Migratory pathway of neural crest progenitors giving rise to the two major pathways of the sympathoadrenal lineage. 19 Figure 3.1 The human adrenal medulla is composed of adrenergic chromaffin cells and NAT is colocalised with adrenergic cells. 109 Figure 3.2 Human phaeochromocytoma tumour samples demonstrate variable PNMT expression 110 Figure 3.3 Variable colocalisation of NAT and PNMT in human phaeochromocytoma tumour samples 111 Figure 3.4 Colocalisation of CGA and NAT in normal human chromaffin cells and in phaeochromocytoma 113 Figure 3.5 Variable expression of NAT in human phaeochromocytoma tumour samples. 117 Figure 3.6 Control sections indicating the specificity of the anti-nat antibody 118 Figure 4.1 Effect of 24-hour Cisplatin pre-treatment on the relative expression of NAT mrna in PC12 cells. 138 Figure 4.2 Effect of 24-hour Cisplatin pre-treatment on NAT staining in PC12 cells 140 Figure 5.1 Proportion of adrenaline and noradrenline in phaeochromocytoma tumour samples from hereditary and non-hereditary patients 155 Figure 5.2 Expression of PNMT mrna in tumour samples from each patient group, and the relationship between tumour PNMT mrna and adrenaline concentrations. 157 Figure 5.3 Expression of NPY mrna and peptide levels in phaeochromocytoma from hereditary and non-hereditary patients, and the relationship between NPY mrna and peptide levels. 158 Figure 5.4 Linear regression analysis demonstrating the relationship between tumour tissue NPY levels (mrna and peptide) and PNMT expression or adrenaline content 160 Figure 5.5 NPY staining in the human adrenal gland 162 Figure 5.6 NPY staining in phaeochromocytoma samples from patients with hereditary and nonhereditary forms of the tumour 163 Figure 6.1 Greater CGA mrna and protein expression in MEN 2 than VHL phaeochromocytoma tumour extracts 176 Figure 6.2 The relationship between catecholamines and CGA mrna and/or protein expression 177 Figure 6.3 CGA Western Blot and Immunohistochemistry 179 Figure 6.4 Relationships between plasma catecholamines (CAT), tumour volume and plasma CGA 182 Figure 7.1 Localization of phaeochromocytoma metastases using MRI and F-FDA 197 Figure 7.2 Intense NPY staining observed in adrenergic chromaffin cells in adrenal glands of balb/c mice. 202 Figure 7.3 Leu-Enk immunoreactivity observed in sub-population of noradrenergic chromaffin cells in adrenal glands of nude mice 203 xvii

18 Figure 7.4 Cytoplasmic expression of NAT in adrenergic chromaffin cells in adrenal glands of balb/c mice 204 Figure 7.5 Intense staining of VMAT1 observed in adrenergic and noradrenergic chromaffin cells, whereas VMAT2 staining was restricted to noradrenergic chromaffin cells in adrenal glands of balb/c nude mice 205 Figure 7.6 Highly variable level of expression of NPY in adrenal phaeochromocytoma from MPNM 209 Figure 7.7 Negligible expression of Leu-Enk in adrenal phaeochromocytoma obtained from an animal generated in MPNM model 210 Figure 7.8 Altered pattern of NAT expression in adrenal phaeochromocytoma from MPNM model, including disappearance of NAT expression in PNMT-positive cells 211 Figure 7.9 Adrenal phaeochromocytoma from MPNM model showing abbarent VMAT2 expression and loss of VMAT1 212 Figure 9.1 Expression of developmental genes in tumour samples from patients with MEN 2 and VHL disease. 271 xviii

19 List of Tables Table 1.1 Conditions mimicking the symptoms of phaeochromocytoma. 56 Table 1.2 Hereditary phaeochromocytomas. 65 Table 2.1 Patient details 84 Table 2.2 List of Primary and Secondary Antibodies used within thesis for immunohistochemistry experiments 94 Table 3.1 Patient information 102 Table 3.2 TH, PNMT and NAT expression in sporadic phaeochromocytoma. 108 Table 3.3 NAT & CGA expression in sporadic phaeochromocytoma. 112 Table 3.4 Expression of NAT & TH in hereditary phaeochromocytomas from VHL and MEN 2 syndrome. 116 Table 4.1 Effects of Cisplatin pre-treatment on NAT mrna expression in PC12 cells 137 Table 4.2 Effects of Cisplatin pre-treatment on NAT functional activity in PC12 cells (DMI; desipramine). 141 Table 5.1 Patient data 151 Table 6.1 Patient information 171 Table 7.1 Gross necropsy findings of nude mice injected with MPC-cells. 198 Table 7.2 The expression of sympathoadrenal markers in chromaffin cells of balb/c and nude mice injected with PBS-only. 201 Table 7.3 Variable expression of the respective sympathoadrenal markers in adrenal phaeochromocytoma of MPNM model 208 xix

20 Abbreviations CGA Chromogranin A ELISA Enzyme linked immunosorbent assay F-FDA 6-[ 18 F]-Fluorodopamine HPLC High performance liquid chromatography I-MIBG Iodo-metabenzylguanidine MEN 2 Multiple Endocrine Neoplasia type 2 MPNM Metastatic phaeochromocytoma nude mouse NAT Noradrenaline transporter NF1 Neurofibromatosis NGF Nerve growth factor NPY Neuropeptide Y PNMT Q-PCR SDHB SDHD TH VHL VMAT Phenylethanolamine N-methyltransferase Quantitative Polymerase Chain Reaction Succinate dehydrogenase subunit B Succinate dehydrogenase subunit D Tyrosine hydroxylase von Hippel-Lindau Vesicular Monoamine Transporter xx

21 Part A The adrenal gland, catecholamines and chromaffin cells I. A brief introduction to the autonomic nervous system The autonomic nervous system is an assortment of efferent nerves linking the central nervous system to the tissues of the body. There are two efferent branches of the autonomic nervous system, which co-ordinate divergent, yet parallel systems necessary to balance the requirements of the inner world in response to stimuli evoked from the outer world. Together, the complementary roles of the sympathetic and parasympathetic nervous system act respectively as the accelerator and breaks of the autonomic nervous system, and provide the checks and balances necessary to regulate internal homeostasis. The sympathetic nervous system is composed of a network of cholinergic preganglionic neurons with regulatory inputs from various brain centres including the rostral ventrolateral medulla, nucleus of the solitary tract and paraventricular nucleus of the hypothalamus. These centres project through the spinal cord to innervate the pre- and para-vertebral sympathetic ganglia, the adrenal glands and enteric or cardiac neuronal networks (Guyenet, 2006). Cholinergic sympathetic efferents synapse with post-ganglionic sympathetic neurons that project to target organs distributed throughout the body, including the heart, kidneys and smooth muscle cells of the vasculature. Following central stimulation, a cascade of events, including the release of acetylcholine from preganglionic nerves, leads to 1