Purinergic Signalling in Skin

PURINERGIC SIGNALLING IN SKIN

AINA VH GREIG MA FRCS

Autonomic Neuroscience Institute Royal Free and University College School of Medicine Rowland Hill Street Hampstead London NW3 2PF

in the

Department of Anatomy and Developmental Biology University College London Gower Street London WCIE 6BT

2002

Thesis Submitted for the Degree of Doctor of Philosophy University of London ProQuest Number: U643205

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT

Purinergic receptors, which bind ATP, are expressed on human cutaneous kératinocytes. Previous work in rat epidermis suggested functional roles of purinergic receptors in the regulation of proliferation, differentiation and apoptosis, for example

P2X5 receptors were expressed on kératinocytes undergoing proliferation and differentiation, while P2X? receptors were associated with apoptosis. In this thesis, the aim was to investigate the expression of purinergic receptors in human normal and pathological skin, where the balance between these processes is changed. A study was made of the expression of purinergic receptor subtypes in human adult and fetal skin. Functional studies using primary human keratinocyte cultures were performed, using specific agonists and antagonists to purinergic receptor subtypes. Purinergic signalling in non-melanoma skin cancer was investigated by immunohistochemical analysis of human basal cell and squamous cell carcinomas. A human cutaneous squamous cell carcinoma cell line (A431) was grown in culture. Specific agonists and antagonists to purinergic receptor subtypes were applied to these cells and changes in cell number were quantified. The expression of purinergic receptors in warts was investigated because of the relationship between human papilloma virus and the development of cutaneous squamous cell carcinomas. Changes in purinergic receptor expression were examined in a rat model of normal and delayed wound healing. The possibility is raised that purinergic signalling within the skin may open the pathway for the development of new treatment modalities for skin cancers, wound healing and perhaps other skin diseases. TABLE OF CONTENTS

ABSTRACT ...... 2

ACKNOWLEDGEMENTS...... 14

PREFACE ...... 15

CH APTER 1 ...... 17

GENERAL INTRODUCTION ...... 17

I. PURINERGIC RECEPTORS AND PURINERGIC SIGNALLING ...... 18

H is t o r y ...... 18

PI RECEPTORS...... 19

P2X RECEPTORS...... 20

P 2Y RECEPTORS...... 24

S ources a n d fate of nucleotides ...... 27 Sources of ATP ...... 28 The purinergic cascade ...... 29 Co-existence of ATP-consuming and ATP-generating pathways ...... 30

Physiological roles of A T P...... 31

S hort -term purinergic sig n a l lin g ...... 31 ATP as a co-transmitter in the nervous system ...... 31 Exocrine and endocrine secretion ...... 31 Regulation of vascular tone ...... 32 Regulation of immune cell function ...... 32

Lo n g -term purinergic s ig n a l l in g ...... 32 Cell proliferation and growth ...... 32 Interactions of purines and pyrimidines with growth factors ...... 33 Purinergic signalling in embryonic development ...... 33 Purinergic signalling in apoptosis and cancer ...... 34

Su m m a r y of features of purinerg ic s ig n a l l in g ...... 39

II. S K I N ...... 40

The e p id e r m is ...... 40 Epidermal architecture ...... 40 Keratinocyte proliferation and stem cells ...... 42 Keratinocyte differentiation ...... 45

3 Agents that ajfect keratinocyte function ...... 47

Ha ir ...... 52 Hair follicle morphogenesis ...... 52 Hair follicle cycle...... 52 Structure and properties of the hair follicle ...... 53

Skin Embryology and Development ...... 57 The epidermis...... 57 Epidermal stratification ...... 57 The periderm ...... 58

Cutaneous wound healing ...... 60 Wound epithelialisation ...... 60 Migrating kératinocytes change phenotype ...... 60 Model of keratinocyte motility ...... 60 Kératinocytes dissect the wound ...... 61 Kératinocytes at the wound margin proliferate ...... 61

Skin CANCER...... 62 Non-melanoma skin cancer ...... 62

Human papilloma virus ...... 66 HPV and human non-melanoma skin cancer ...... 67 III. ROLE OF PURINES IN SKIN...... 69

Short term purinergic signalling ...... 69 Purinergic signalling in cutaneous blood vessels ...... 69

Long term purinergic signalling ...... 69 Purinergic signalling in the epidermis ...... 69 Purinergic signalling in cutaneous wound healing ...... 71

Project aims ...... 73

RESULTS...... 74

SECTION A ...... 75

NORMAL TISSUES...... 75

CHAPTER 2 ...... 76

PURINERGIC RECEPTORS ARE PART OF A FUNCTIONAL SIGNALLING SYSTEM FOR PROLIFERATION AND DIFFERENTIATION OF NORMAL HUMAN EPIDERMAL KERATINOCYTES...... 76 ABSTRACT...... 77 INTRODUCTION...... 78 MATERIALS AND METHODS...... 80 Tissues ...... 80 Preparation of antibodies ...... 80 Immunohistochemistry ...... 80 Double-labelling techniques ...... 82 Photography ...... 83 Human primary cultures of kératinocytes in low calcium medium ...... 84 Proliferation Assay ...... 84 Statistical analysis ...... 85 Subcutaneous injection experiment ...... 85

RESULTS...... 86 P2Xs and P2Xj receptors are expressed in human skin ...... 86 P2Y] and P2Y2 receptors are expressed in human skin ...... 87 Cells positive for P2Yj and P2Y2 receptors also express markers for cellular proliferation ...... 87 Cells positive for P2Xs receptors also express markers for keratinocyte differentiation ...... 87 Double-labelling ofP2Xy receptors with markers for apoptosis in human skin show co-localisation ...... 87 Control immunostaining experiments ...... 88 Functional studies of primary keratinocyte cultures show that purinergic receptor agonists can alter cell number...... 88 Direct subcutaneous injection of purinergic receptor agonists results in changes in the morphology of the rat epidermis ...... 89 DISCUSSION...... 90 FIGURE LEGENDS...... 94

CHAPTER 3 ...... n o

PURINERGIC RECEPTORS ARE PART OF A SIGNALLING SYSTEM FOR PROLIFERATION AND DIFFERENTIATION IN DISTINCT CELL LINEAGES IN HUMAN ANAGEN HAIR FOLLICLES...... 110

ABSTRACT...... I l l INTRODUCTION...... 112 MATERIALS AND METHODS...... 114 Tissues ...... 114 Antibodies ...... 114 Immunohistochemistry ...... 114 Double-labelling techniques ...... 115 Photography ...... 116 RESULTS...... 117 P2Xs, P2Xy, P2Y] and P2Y2 receptors were expressed in human anagen hair follicles...... 117 Outer root sheath cells positive for P2Y] receptors, also expressed markers for cellular proliferation. Cortex and medulla cells positive for P2Y2 receptors, did not express markers for proliferation. Double-labelling of P2Xs receptors with involucrin showed co-localisation in the inner root sheath and in cells at the edge of the medulla ...... 117 Control immuno staining experiments ...... 118 DISCUSSION...... 119 FIGURE LEGENDS...... 122

CHAPTER 4 ...... 128

PURINERGIC SIGNALLING IN HUMAN FETAL SKIN ...... 128

ABSTRACT...... 129 INTRODUCTION...... 130 MATERIALS AND METHODS...... 133 Tissues ...... 133 Antibodies ...... 133 Immunohistochemistry ...... 133 Double-labelling techniques ...... 134 Photography ...... 136 RESULTS...... 137 P2Xs^ P2Xy_ P2Y] and P2Y2 receptor expression in human fetal epidermis 137 Comparison of receptor distribution in fetal and adult epidermis ...... 137 Double-labelling ofP2Yj receptors with markers for cellular proliferation 138 Double-labelling of P2Xs receptors with markers for keratinocyte differentiation.

...... Double-labelling of P2Xy receptors with markers for apoptosis in fetal epidermis show co-localisation ...... 138 DISCUSSION...... 139 FIGURE LEGENDS...... 143

SECTION B ...... 149

PATHOLOGICAL TISSUES...... 149

CHAPTER 5 ...... 150

PURINERGIC RECEPTOR EXPRESSION IN THE REGENERATING EPIDERMIS IN A RAT MODEL OF NORMAL AND DELAYED WOUND HEALING...... 150

ABSTRACT...... 151 INTRODUCTION...... 152 MATERIALS AND METHODS...... 154 Study design ...... 154 Surgical technique ...... 154 Nerve growth factor (NGF) - treated flaps ...... 156 Preparation of primary antibodies ...... 156 Immunohistochemistry ...... 157 Optical densitometry ...... 158 RESULTS...... 159 DISCUSSION...... 161 FIGURE LEGENDS...... 165 TABLE LEGENDS...... 176

CHAPTER 6 ...... 177

PURINERGIC SIGNALLING IN NON-MELANOMA SKIN CANCER...... 177

ABSTRACT...... 178 INTRODUCTION...... 179 MATERIALS AND METHODS...... 181 Tissues ...... 181 Preparation of antibodies ...... 181 Immunohistochemical methods for frozen sections ...... 182 Immunohistochemical method for paraffin sections ...... 184 Photography ...... 186 A431 cell line ...... 186

1 Proliferation Assay ...... 187 Statistical analysis ...... 188 RESULTS...... 189

PlXs, P2Xj, P2Y], P2Y2 and P 2 Y4 receptors were expressed in frozen sections of basal cell carcinomas ...... 189

P2Xs, P2Xy, P2Y] and P 2 Y2 receptors were expressed in frozen sections of squamous cell carcinomas ...... 189 P2Xs and P2Xy receptors were expressed in paraffin sections of basal cell and squamous cell carcinomas ...... 190

P2Xs, P2Xj, P2Y] and P 2 Y2 receptors were expressed in A431 cells grown in culture ...... 192 Actions of purinergic receptor agonists on cell numbers of cultured cutaneous squamous cell carcinoma, A431 cells ...... 192 DISCUSSION...... 194 FIGURE LEGENDS...... 198 TABLE LEGENDS...... 217

CHAPTER 7 ...... 220

P2X5 AND P2Xy RECEPTORS IN HUMAN WARTS AND CIN-612 RAFT CULTURES OF HUMAN PAPILLOMAVIRUS INFECTED KERATINOCYTES. 220

ABSTRACT...... 221 INTRODUCTION...... 222 MATERIALS AND METHODS...... 224 Tissues ...... 224 Raft organotypic cultures of differentiated HPV infected kératinocytes ...... 224 Antibodies ...... 224 Immunohistochemical method for paraffin sections ...... 224 Photography ...... 225 RESULTS...... 226 P2Xs and P2Xj receptors in paraffin sections of normal human skin ...... 226

P2 X5 and P2Xj receptors in paraffin sections of human warts ...... 226 P2Xs and P2Xy receptors in paraffin sections of raft cultures of normal human kératinocytes and ofClN612 (HPV 31) cells...... 227 DISCUSSION...... 228 FIGURE LEGENDS...... 230 CHAPTER 8 ...... 236

GENERAL DISCUSSION...... 236

1. M ajor fin d ing s of this t h e s is ...... 237

2. Lo n g term trophic roles of purines a n d pyrimidines ...... 243

3. Purinerg ic sig nalling in w o u n d h e a l in g ...... 246

4. Pu rinerg ic sig nalling a n d therapeutic applications ...... 250 Therapeutic potential of purinergic signalling in general ...... 250 Therapeutic potential of purinergic signalling in skin disease ...... 253

REFERENCES...... 255

APPENDIX...... 301

Appen d ix - Gen er a l T issu e C u l t u r e ...... 302

t a b l e o f f ig u r e s

Figure 1.1: Structure of P2X receptors ...... 21 Figure 1.2: Structure of P2Y receptors ...... 24 Figure 1.3: The skin and its appendages ...... 41 Figure 1.4: Schematic interpretation of the effects of c-Myc and p-catenin on the epidermal stem cell compartment in culture ...... 44 Figure 1.5: Follicular morphogenesis and hair cycling ...... 53 Figure 1.6: Human hair follicle structure ...... 54 Figure 1.7: Hair follicle lineages ...... 55 Figure 2.1: Human epidermal kératinocytes in frozen sections of leg skin expressed P2Xs and P2X? receptors ...... 94 Figure 2.2: Human epidermal kératinocytes from different regions of the body also expressed P2Xs and P2X? receptors in a similar fashion to that seen in leg skin in Fig 2.1 ...... 96

Figure 2.3: Human epidermal kératinocytes from leg skin expressed P2Yi and P 2 Y2 receptors ...... 98

Figure 2.4: Double-labelling of P2Yi and P 2Y2 receptors with markers of proliferation showed co-localisation within a subpopulation of basal and parabasal kératinocytes. Double-labelling of P2Xg receptors with markers

9 of differentiated kératinocytes showed co-localisation within the stratum spinosum, and double-labelling of P2X? receptors with markers of apoptosis in human leg skin showed co-localisation within the stratum

comeum ...... 100 Figure 2.5: At 48 hours after application to primary keratinocyte cultures: a) The effect of 100-5000pM ATP was significantly (P<0.001) more potent than

that of the same dose of adenosine, b) 30)liM 8 -(p-

sulfophenyl)theophylline ( 8 /?SPT) caused no significant block of the effect of adenosine, c) 30pM Dipyridamole (Dip) had no significant effect

on the response to adenosine...... 102 Figure 2.6: At 48 hours after application of ATP to primary human keratinocyte

cultures with either a) 30|xM 8 -(p-sulfophenyl)theophylline ( 8 pSPT) or b) 30|iM Dipyridamole (Dip), no significant effect on the response to ATP was observed ...... 103 Figure 2.7: At 48 hours after application, ATP (1-lOpM) and UTP (lOOpM) caused an increase in cell number of primary human keratinocyte cultures, whereas ATPyS (lOO-SOOpM) and ATP (lOOpM) caused a significant decrease...... 104 Figure 2.8: At 48 hours after application, 2MeSADP (500pM) caused a significant increase in cell number of primary human keratinocyte cultures...... 105 Figure 2.9: At 48 hours after application, BzATP (100-500pM) caused a significant decrease in cell number of primary human keratinocyte cultures...... 106 Figure 2.10: At 48 hours after application of ATP with lOOpM suramin, suramin

caused a significant block in the effect of lOOfxM ATP (*P<0.01) and

1000|uiM ATP (**P<0.001) on cell number of primary human keratinocyte cultures...... 107 Figure 2.11: Subcutaneous injection of purinergic receptor agonists caused changes in the thickness of the epidermis of rat skin ...... 108 Figure 3.1: Expression of P2Xs and P2X? receptors in human anagen hair follicles... 122

Figure 3.2: Expression of P2Yi and P 2Y2 receptors in human anagen hair follicles... 124

Figure 3.3: Double-labelling of P2Y% and P 2 Y2 receptors with markers for cellular

proliferation and double-labelling of P 2X5 receptors with markers for keratinocyte differentiation in anagen hair follicles ...... 126

Figure 4.1: P2Xs, P2X?, P2Yi and P 2 Y2 receptors were expressed in human fetal epidermis ...... 143

10 Figure 4.2: P 2X5, P2X?, P2Y1 and P2 Y2 receptor expression in adult human epidermis. 145 Figure 4.3: Double-labelling of P2Yi and P2Yz receptors with markers for cellular

proliferation. Double-labelling of P 2X5 receptors with markers for keratinocyte differentiation. Double-labelling of P2Xv receptors with markers for apoptosis ...... 147 Figure 5.1: Comparison of P2Xs receptor immunostaining in the epidermis of normal skin, and in the regenerating epidermal wound edge of innervated and denervated excisional wounds and innervated wounds treated with NGF. 165 Figure 5.2: Histogram showing the optical density (arbitrary units) of P2Xs receptor immunostaining in epidermal kératinocytes of different preparations (listed top right) in order to quantify the amount of immunostaining 167 Figure 5.3: Comparison of P2X? receptor immunostaining in the epidermis of normal skin, and in the regenerating epidermal wound edge of innervated and denervated excisional wounds ...... 168 Figure 5.4: Comparison of P2Y] receptor immunostaining in the epidermis of normal skin, and in the regenerating epidermal wound edge of innervated and denervated excisional wounds and in both innervated and denervated wounds treated with NGF ...... 170 Figure 5.5: Histogram showing the optical density (arbitrary units) of P2Yi receptor immunostaining in epidermal kératinocytes of the different preparations (listed top right) in order to quantify the amount of immunostaining ...... 172

Figure 5.6: Comparison of P 2 Y2 receptor immunostaining in the epidermis of normal skin, and in the regenerating epidermal wound edge of innervated and denervated excisional wounds and in both innervated and denervated wounds treated with NGF ...... 173

Figure 5.7: Histogram showing the optical density (arbitrary units) of P 2 Y2 receptor immunostaining in the epidermal kératinocytes of different preparations (listed top right) in order to quantify the amount of immunostaining ...... 175 Figure 6.1: P2Xs and P2X? receptor expression in basal cell carcinoma ...... 198

Figure 6.2: P2Yi, P 2Y2 and P2Y4 receptor expression in basal cell carcinoma ...... 200

Figure 6.3: P 2X5, P2X?, P2Yi and P2Y2 receptor expression in squamous cell

carcinoma ...... 202 Figure 6.4: P2Xs and P2Xv receptors were expressed in paraffin sections of different subtypes of basal cell carcinoma; sections counterstained with haematoxylin to stain nuclei blue ...... 204

11 Figure 6.5: P2Xs and P2X? receptors were expressed in paraffin sections of different subtypes of squamous cell carcinoma; sections counterstained with haematoxylin to stain nuclei blue ...... 206

Figure 6 .6 : P2X5, P2X?, P2Yi and P2 Y2 receptor expression in squamous cell carcinoma, A431 cells grown in culture ...... 208 Figure 6.7: Change in cell number of squamous cell carcinoma, A431 cells at 48 hours after ATP application ...... 210

30pM 8 -(p-sulfophenyl)theophylline ( 8 /?SPT) caused no significant block of the effect of adenosine, c) 30pM Dipyridamole (Dip) caused no

significant effect on the adenosine response ...... 212 Figure 6.9: At 48 hours after application of either a) 30pM S-(p-

sulfophenyl)theophylline ( 8 /?SPT) or b) 30pM Dipyridamole (Dip), no significant effect on the response to ATP was seen on A431 cell number.213 Figure 6.10: Comparison of effect of ATP with UTP and ATPyS at 48 hours after drug application to A431 cells ...... 214 Figure 6.11: Percentage change in A431 cell number with BzATP, P2X? receptor subtype agonist, at 48 hours after drug application ...... 215 Figure 6.12: Percentage change in A431 cell number with 2MeSADP, P2Yi receptor subtype agonist, at 48 hours after drug application ...... 216 Figure 7.1: Expression of P2Xs and P2X? receptors in paraffin sections of normal human skin. Nuclei were counterstained blue with haematoxylin 230 Figure 7.2: Expression of P2Xs and P2X? receptors in paraffin sections of human warts. Nuclei were counterstained blue with haematoxylin...... 232 Figure 7.3: Expression of P2Xs and P2X? receptors in paraffin sections of raft cultures of normal human kératinocytes and of CIN612 (HPV 31) cells. Nuclei were counterstained blue with haematoxylin ...... 234

12 TABLE OF TABLES

Table 1.1: Summary of properties of P2X receptor subtypes ...... 22 Table 1.2: Summary of properties of P2Y receptor subtypes ...... 26 Table 1.3: Role of various agents in regulating keratinocyte function ...... 47

Table 1.4: HPV types and their associated disease states ...... 66 Table 5.1: Summary of purinergic receptor subtype expression in epidermal kératinocytes of normal control skin and in the regenerating epidermis of: control wounds, NGF-treated control wounds, denervated wounds and NGF-treated denervated wounds ...... 176 Table 6.1: Distribution of P2X and P2Y receptor subtypes in normal skin, BCCs and SCCs...... 217

Table 6.2: Analysis of P2 X5 and P2X? receptor distribution in paraffin sections of basal cell carcinoma subgroups ...... 218 Table 6.3: Analysis of P2Xs and P2X? receptor distribution in paraffin sections of squamous cell carcinoma subgroups ...... 219

LIST OF ABBREVIATIONS

AA - amino acids HPV - human papillomavirus ABC - avidin biotin complex 2MeSADP - 2-methylthioadenosine 5’- ADP - adenosine 5’-diphosphate diphosphate ATP - adenosine 5’-triphosphate NaCl - sodium chloride ATPyS - adenosine 5’-0-(3- NGF - nerve growth factor thiotriphosphate) NGS - normal goat serum BCG - basal cell carcinoma NHEK - normal human epidermal BzATP - 2’-& 3’-0-(4-benzoyl-benzoyl) kératinocytes adenosine 5’-triphosphate NHS — normal horse serum DAB - nickel-diaminobenzidine PBS - phosphate buffered saline DMEM - Dulbecco’s Modified Eagles’ PCNA - proliferating cell nuclear Medium antigen DMSO- di-methyl sulfoxide SCC - squamous cell carcinoma EDTA - ethylenediaminetetra-acetic TUNEL - TdT-mediated dUTP nick end acid labelling PCS - fetal calf serum UTP - uridine 5’-triphosphate

13 ACKNOWLEDGEMENTS

I am grateful to the many people who have contributed to this thesis. My first thanks go to my two supervisors, Professor Bumstock and Professor McGrouther, who both provided excellent guidance and support. I would also like to thank present and former members of the Autonomic Neuroscience Institute for teaching me, for their support and for helpful discussions: Ute Groschel-Stewart, Michelle Bardini, Tim Robson, Eamonn Moules, Gill Knight, Philippe Bodin, Astrid Hoebertz, Mina Ryten, Alex Cheung, Annie Evans and Chrystalla Orphanides amongst many others. I would also like to thank Dr Malcolm Rustin in the Department of Dermatology, Royal Free Hospital. I would like to thank those with whom I worked at the RAFT Institute of Plastic Surgery, Mount Vernon Hospital, where I did my cell culture experiments. I especially would like to thank Dr Claire Linge and Jamie Shelton for teaching me keratinocyte culture and for fruitful discussions and advice, and Dr Alison Cambrey for providing me with fetal tissue. Philip Lim and Claire Linge, from RAFT provided archive tissue in paraffin section of non-melanoma skin cancers. Dr Voumeen Healy, Department of Pathology, Royal Marsden Hospital, helped with analysis of the archive samples of non melanoma skin cancer. Scott Cuthill from Roche Welwyn provided archive tissue in paraffin section of warts and raft organotypic cultures of human foreskin kératinocytes and CIN-612 cells. I would also like to thank Roche Bioscience, Palo Alto, California, USA, who provided P2Xs and P2X? receptor antibodies. I would also like to thank those with whom I worked in collaboration. Stuart James from the Blond Mclndoe Centre, Department of Surgery, Royal Free and University College Medical School, performed the surgery in the rat model of wound healing. Liz Clayton from RAFT developed the method for purinergic receptor staining in paraffin sections. I would like to thank Mike, my family and all my friends for their support and encouragement. I was awarded a Research Fellowship from The Wellcome Trust, The Simpson Surgical Research Fellowship from The Royal College of Surgeons of. England and a Research Studentship from the Anatomy Department, UCL. I would like to thank those organisations for funding my research.

14 PREFACE

In this preface I would like to give a short outline of the different chapters in this thesis. The work presented here has focussed on purinergic signalling in epidermal kératinocytes under both normal and pathophysiological conditions. Extracellular nucleotides are now recognised as important signalling molecules mediating a wide range of functions. They act via two types of receptors: P2X receptors, which are ligand-gated ion channels, and P2Y receptors, which are G-protein coupled receptors. The aim of my thesis, to study purinergic signalling in human skin, was provoked by a previous immunohistochemical study on the skin of rats suggesting the presence of P2 receptors on epidermal kératinocytes, and some brief pharmacological reports that ATP had an effect on growth, proliferation and terminal differentiation in epidermal kératinocytes. Chapter 1 provides a General Introduction: (I) to the rapidly expanding field of purinergic signalling, introducing the history, classification and characterisation of receptor subtypes, the source, breakdown and physiological roles of extracellular nucleotides; ( 2 ) to epidermal and keratinocyte biology, hair follicles, skin embryology and development, cutaneous wound healing as applied to the regenerating epidermis, skin cancers and human papilloma viruses; (3) to the current knowledge about purinergic signalling in the skin. In this thesis the expression of P2 receptors was shown first of all in normal tissues (Section A), including in normal human adult skin (Chapter 2), in anagen hair follicles (Chapter 3), and in human fetal skin (Chapter 4). The expression of P2 receptors was also shown in a variety of pathological tissues (Section B), including in the regenerating epidermis in a rat model of normal and delayed wound healing

(Chapter 5), non-melanoma skin cancers (Chapter 6 ), and in warts and HPV infected raft organotypic cultures (Chapter 7). In the first experimental chapter (Chapter 2), the expression of P2 receptor subtypes was studied in frozen sections of human skin using immunohistochemistry and functional experiments on primary cultures of human epidermal kératinocytes were performed where purinergic receptor subtype agonists were applied to cells and changes in cell number were quantified via a colorimetric assay. Evidence is presented that P2 receptors are expressed in spatially distinct zones of the human epidermis and that they

15 have functional roles in keratinocyte proliferation, differentiation and terminal differentiation/apoptosis. In Chapter 3, the expression of P2 receptors was studied in frozen sections of human anagen hair follicles using immunohistochemistry and double labelling techniques with markers of proliferation, differentiation and apoptosis. In Chapter 4, the expression of P2 receptors was studied in frozen sections of human fetal epidermis using immunohistochemistry and double labelling techniques with markers of proliferation, differentiation and apoptosis. Chapter 5, investigated the changes in the expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates are normalised. Changes in the level of receptor expression were quantified using optical density measurements of immunopositive epidermal kératinocytes.

In Chapter 6 , immunohistochemical analysis of frozen sections in human basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) for P2 receptors was performed, accompanied by detailed analysis of archive material of tumour subtypes in paraffin sections. Functional studies were performed using a human cutaneous SCC cell line (A431), where purinergic receptor subtype agonists were applied to cells and changes in cell number were quantified via a colorimetric assay. Chapter 7, explored purinergic receptor staining in cutaneous warts and in raft organotypic cultures of human foreskin kératinocytes and a human papilloma virus infected cell line, CIN-612. A detailed discussion of results is provided at the end of each experimental chapter. In the General Discussion (Chapter 8 ), the major findings of this thesis will be summarised and discussed in terms of the advantages and disadvantages of the techniques used and possible directions for future research. Broader issues will be discussed that have been raised by the detailed discussion of results in the experimental chapters, including the long-term trophic roles of purines and pyrimidines, purinergic signalling in wound healing and lastly the therapeutic potential of purinergic agonists and antagonists in skin diseases and other pathological conditions.

16 CHAPTER 1 GENERAL INTRODUCTION

17 With the increasing evidence for physiological roles for purinergic receptors, the main aim of this thesis was to establish whether these receptors have a functional role in the homeostasis, tissue regeneration, fetal development and pathophysiology of the epidermis of human skin. This general introduction will give a brief review of current knowledge about purinergic signalling, epidermal biology and previously known roles of purines in the skin.

I. PURINERGIC RECEPTORS AND PURINERGIC SIGNALLING

History

In 1929, adenosine 5’-triphosphate (ATP), a molecule belonging to the purine family, was identified in muscle extracts (Fiske and Subbarow 1929; Lohmann 1929). At first this bio-molecule was thought to be primarily concerned with muscle contraction. It was then demonstrated that ATP was generated during the breakdown of glucose to lactic acid (anaerobic glycolysis), and via the citric acid cycle and aerobic oxidation (oxidative phosphorylation) in mitochondria. The recognition that ATP had a central role in energy exchanges in biological systems allowed Lippman to introduce his general hypothesis for energy transfer in living cells (Lippman 1941). It became apparent that purines were also able to exert profound extracellular effects on different organs and tissues. Drury and Szent-Gyorgyi were the first to propose the concept of purines as extracellular signalling molecules. They showed that adenosine and adenosine 5 ’ -monophosphate (AMP) had biological effects on the mammalian heart (Drury and Szent-Gyorgyi 1929). There was considerable resistance to the concept that ATP could exert any extracellular physiological action because of the discovery of the essential intracellular role as a universal source of chemical energy for all living cells. This line of thought was reinforced by the fact that a molecule of the size and charge of ATP could not cross the plasma membrane by simple diffusion (Glynn 1968; Chaudry 1982). However, many studies followed, 'confirming the extracellular role of nucleotides in the cardiovascular system. A paper demonstrating different actions of adenosine and ATP on vasodilatation, hypotension and ileum contraction, indicated the existence of multiple purine receptors for the first time (Gillespie 1934).

18 In 1972, Bumstock proposed purinergic neurotransmission for the first time (Bumstock 1972), suggesting that ATP was a transmitter involved in non-adrenergic, non-cholinergic nerve-mediated responses of smooth muscle in the gastrointestinal tract and in the bladder. In 1976, the concept of co-transmission was introduced (Bumstock 1976). However, both concepts have only been generally accepted over the last ten years and extracellular purines and pyrimidines have since been implicated in a wide range of biological processes, including smooth muscle contraction, exocrine and endocrine secretion, inflammation, platelet aggregation and pain, amongst many others (Abbracchio and Bumstock 1998; Ralevic and Bumstock 1998). The term ‘purinergic receptors’ was first formally introduced in 1978 (Bumstock 1978). Receptors were divided into ‘Pi-purinoreceptors’, with adenosine as main ligand, and ‘P2-purinoreceptors’, with adenosine 5’-diphosphate (ADP) and ATP as main ligands. In principal, this classification remains true, however, receptors for pyrimidines are now included in the P2-receptor family (Fredholm et al. 1994). In 1985, Bumstock and Kennedy proposed a basis for distinguishing between two types of P2 receptors, P2X and P2Y, based largely on pharmacological criteria (Bumstock and Kennedy 1985). Based on cloning, second messenger systems and pharmacology of P2 receptors, a new nomenclature system was proposed (Abbracchio and Bumstock 1994): P2 receptors are divided into two families: P2X ionotropic ligand-gated ion-channel receptors and P2Y metabotropic G-protein-coupled receptors.

PI receptors

Four members of the adenosine/Pl receptor family have now been cloned and characterised from a variety of species: Ai, A 2A, A2B, A 3 , and, with the exception of the

A2A subtype, selective agonists and antagonists have been identified. All PI receptors couple to G proteins, and modulate adenylate cyclase activity in an inhibitory (Ai, A 3 ) or stimulatory (A 2A, A2b) fashion, resulting in cyclic AMP (cAMP) changes. However, they show distinct tissue distributions and pharmacological profiles. Adenosine plays a major role as a vasodilator in the heart and as a neuromodulator acting as a general central nervous system (CNS) depressant, e.g. the stimulatory effects of caffeine and theophylline are produced by inhibition of adenosine actions. Many diseases have been envisioned as candidates for treatment with compounds acting upon the adenosine regulatory system, including asthma, Parkinson’s disease and psychiatric disorders. Since this thesis will focus on the role of members of the P2 receptor family, the reader is referred to recent reviews for a summary of the distribution, pharmacology and

19 physiology of PI receptors (Ralevic and Bumstock 1998; Fredholm et al. 2000a; Fredholm et al. 2000b).

P2X receptors

Seven mammalian members of the family of P2X receptors have been cloned so far (P2Xi - P2X?). The P2Xi.7 subunits share an overall sequence identity, ranging from 35% to 48%, and their sizes range from 379 to 595 AA. The p2xg receptor subtype has also been cloned from embryonic chick skeletal muscle (Bo et al. 2000). P2X receptors define a novel class of ligand-gated cation channels (Fig 1.1), consisting of two transmembrane domains, separated by an extensive A-glycosylated extracellular loop, always containing 10 cysteine residues, and short intracellular amino (N)-and carboxy

(C)-termini, with the exception of P 2X2 and P2X?, which have a more extended C- terminal tail. ATP represents the major ligand and induces rapid and selective permeability to cations (Na^, K^, Ca^^), explaining their abundant distribution on excitable cells such as neurons, smooth muscle and glial cells, and their role as mediators of fast excitatory neurotransmission in both the central and peripheral nervous system. Although all P2X receptors mediate fast signalling, their localisation, function and pharmacological characteristics are different. Based on agonist efficacy and desensitisation characteristics, P2X receptors have been grouped into three distinct classes (Dubyak et al. 1996). Group 1 includes P2X% and P2Xs receptors with a high affinity for ATP (EC5o= IpM) that are rapidly activated and desensitised. Group 2 includes P2Xz, P2 X4, P2Xg and P2Xô receptors that have a lower affinity for ATP

(EC5o= lOpM), show slow desensitisation and sustained depolarising currents. Group 3 is represented by the P2X? receptor that has very low affinity for ATP (EC 5o= 300- 400p,M), shows little or no desensitisation, and in addition to functioning as an ATP- gated ion channel can also function as a non-selective ion pore (Di Virgilio et al. 1999).

20 A P2X receptor subunit B Multimeric P2X channel

Cations

Extracellular

Intracellular

NHz COOH

Figure 1.1: Structure of P2X receptors A) P2X receptor subunits consist of two hydrophobic transmembrane domains, a large A-glycosylated (triangles) extracellular loop, and intracellular N- and C-termini. The extracellular loop includes two to six potential A-linked glycosylation sites and 10 conserved cysteine residues, which may form intramolecular disulfide bridges (-S-S-). B) At least three (or four) subunits are thought to form a functional P2X channel, allowing passage of cations.

Functional P2X channels are homomultimers or heteromultimers formed by the association of at least three subunits. For a summary of possible P2X subunit combinations, see Torres et al (1999). It is not yet known how the properties of different P2X subunits influence the phenotype of the heteromultimeric receptors. Alternative splicing and species-differences may increase heterogeneity of P2X receptors. The following table (Table 1.1) will give a brief overview of distribution and most important characteristics of each receptor subtype.

21 Table 1.1: Summary of properties ofP2X receptor subtypes Receptor Site Agonist Antagonist Receptor properties

P2Xi Smooth muscle a,P-meATP TNP-ATP, rapidly (bladder, vas deferens, isoPPADS desensitising smooth muscle layers of small arteries) P2X2 Nervous tissues, bladder and Weak isoPPADS, significant intestine agonists: Reactive permeability 2MeSATP, Blue 2 to p,y-meATP, sensitive to ATPyS extracellular acidification P2X3 Nociceptive sensory neurons a,P-MeATP TNP-ATP, rapidly (trigeminal, nodose and isoPPADS desensitising dorsal root ganglia), endothelial and epithelial cells P2 X4 Generally distributed ATP TNP-ATP slowly throughout the body and desensitising found extensively in the brain, acinar cells of salivary glands P2Xs Brain, heart, spinal cord, ATPyS PPADS, Non adrenal medulla, thymus and Suramin desensitising lymphocytes, proliferating (non- and differentiating cells in selective) keratinised and non- keratinised epithelia and in growing hair follicles, developing chick and rat skeletal muscle, skeletal muscle differentiation P2X6 CNS, but also present in 2MeSATP none Minimal thymic epithelial cells, desensitising developing chick and rat skeletal muscle P2X? Haematopoietic BzATP KN-62, Bifunctional: (macrophages, monocytes, Coomassie either cation lymphocytes, granulocytes) Brilliant channel or and nervous tissues Blue G forms a large (microglia), keratinised pore and epithelium allows calcium entry and cell death

22 Two P2X receptor subtypes are of particular interest in this thesis, and are discussed below:

P2Xsreceptor Transcripts for this receptor have been identified in brain, heart, spinal cord, adrenal medulla, thymus and lymphocytes (Collo et al. 1996; Garcia-Guzman et al. 1996; Le et al. 1997). There is clear evidence of a P2Xi/s heteromeric receptor with properties distinct from P2X% and P2Xs receptors (Torres et al. 1998; Haines et al. 1999a; Le et al. 1999). Protein expression of P2Xs receptors has been demonstrated in the proliferating and differentiating cell layers in keratinised and non-keratinised rat epithelia and in growing hair follicles, implying a role in proliferation and/or differentiation (Groschel-Stewart et al. 1999a; Bardini et al. 2000). Additionally, work from our lab has recently reported a time-course dependent expression in developing chick and rat skeletal muscle (Meyer et al. 1999) with possible roles in myotube formation (Ryten et al. 2001). ATP has been shown to regulate the differentiation of rat skeletal muscle cells by activation of P2Xs receptors on satellite cells (Ryten et al.

2 0 0 2 ). P2X5 receptors have also been implicated in the regulation of osteoblastic differentiation and proliferation (Hoebertz et al. 2000). The order of agonist potencies at the recombinant rat receptor is ATPyS> ATP> 2MeSATP> apmeATP> ADP (Khakh et al. 2000). There are no selective antagonists for the P2Xg receptor. Non-selective antagonists include suramin and PPADS (Khakh et al. 2000).

P2X7 receptor The P2X? receptor, was previously called the P2Z receptor and cloned from rat macrophages and brain in 1996 (Surprenant et al. 1996). The receptor is found in haematopoietic (macrophages, monocytes, lymphocytes, granulocytes) and nervous tissues (microglia) (Collo et al. 1997). P2X? receptors are associated with terminally differentiated kératinocytes in the stratum comeum of the rat epidermis, which have undergone a specialised form of apoptosis (Groschel-Stewart et al. 1999a). It is a bi functional molecule that functions not only as a selectively permeable cation channel under physiological conditions, but also, upon prolonged agonist stimulation and low levels of divalent cations, as a cytolytic pore permeable to larger hydrophilic molecules up to 900Da as well as to ions (Rassendren et al. 1997; Virginio et al. 1997). Since the latter function is associated with cytotoxicity, the receptor has been connected to lytic and apoptotic events, and there is increasing evidence that this process is dependent on the caspase signalling cascade (Coutinho-Silva et al. 1999; Ferrari et al. 1999). P2Xv

23 receptors on macrophages can trigger release of the pro-inflammatory cytokines D L-lp

and IL-6 , and may have a role in immunomodulation and inflammation (Ferrari et al. 1997a). P2Xv fails to form heteromultimers with other subunits and reacts preferentially with ATP^' and benzoyl ATP. The order of agonist potency at human lymphocytes is BzATP> ATP> 2MeSATP> ATPyS (Khakh et al. 2000). Potent antagonists of P2X? receptors include Coomassie Brilliant Blue G (Jiang et al. 2000) and isoquinolines like 1 -[N, 0-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), which is more potent in human P2X? receptors than in rat (Humphreys et al. 1998).

P2Y receptors

The P2Y family of receptors is a novel subclass of the superfamily of G protein- coupled receptors, each having seven transmembrane domains (Fig 1.2). The third intracellular loop and the C-terminus are thought to be involved in G protein-coupling, whereas the third, sixth and seventh transmembrane domains have been implicated in nucleotide binding (Jacobson et al. 1999).

P2Y receptor

Extracellular

Intracellular

COOH

Figure 1.2: Structure of P2Y receptors P2Y receptors consist of seven transmembrane domains, with an extracellular N- terminus and an intracellular C-terminus.

P2Y receptors are widely distributed throughout the body and exhibit more sequence diversity than any other known family of G protein-coupled receptors. Most subtypes couple through G proteins to phospholipase C (PLC), mainly to PLCp, and subsequent formation of inositol 1,4,5-triphosphate (IP 3) and diacylglycerol (DAG), but

24 inhibition of adenylyl cyclase (AC) can also occur (Boyer et al. 1993; Boyer et al. 1994; Feolde et al. 1995). P2Yn receptors can activate adenylyl cyclase and contribute to the formation of cyclic AMP (Torres et al. 2002). IP 3 activates IPg-sensitive channels on the endoplasmic reticulum, leading to transient elevation of cytosolic free Ca^"^ ([Ca^"^],); both DAG and elevated [Ca^^Ji activate certain forms of protein kinase C (PKC), followed by further downstream signalling events. Recombinant P2Y receptors affect a narrow range of signalling pathways (PLC, AC), whereas endogenous receptors affect a much wider range of intracellular signalling cascades, including phospholipase A 2

(PLA2), phospholipase D (PLD), mitogen activated protein kinase (MAPK), in addition to PLC and AC (King et al. 2000).

Seven mammalian P2Y receptors have been cloned to date: P2Y%, P 2 Y2, P2Y4,

P2Yô, P2Yii, P2Y i2 and P2Yn (King et al. 2000; Communi et al. 2001) (see Table 1.2). The lower case, p2y, is used for mammalian orphan receptors or functional non mammalian receptor proteins without a mammalian homologue. The chick p2ys receptor may be the homologue of the human P 2 Yô receptor, whereas a mammalian homologue of the Xenopus p2yg has not yet been identified. The putative p2ys, p2y?, p2yg and p2yio receptors turned out not to be ‘true’ members of the P2Y receptor family: p2 y? was found to be identical with the leukotriene B 4 receptor and the others, when expressed in appropriate cell lines with no intrinsic purinoreceptors, lost their sensitivity to ATP (Ralevic and Bumstock 1998; King et al. 2000).

25 Table 1.2: Summary of properties ofP2Y receptor subtypes Receptor Site Agonist Antagonist Receptor properties P2Yi Vascular endothelial ADP, ATP, MRS2179 Platelet aggregation, cells, connective, 2MeSADP vascular relaxation, immune and neural endothelial cell tissues proliferation. P2Y2 Epithelial cells, UTPyS, PPADS, Epithelial Cl endothelial vascular UTP, ATP Suramin secretion, smooth muscle cells vascular relaxation, keratinocyte proliferation. P2Y4 Placenta UTP, ATP PPADS Not established P2Y6 Thymus, lung, spleen UDP, UTP PPADS, Not established and smooth muscle Suramin P2Yii Spleen and ATP Suramin Not established granulocytes P2Y i2 Platelets, and to a ADP Suramin Platelet aggregation smaller extent in brain P2Y i3 Spleen and brain ADP, Not known Stimulation of 2MeSADP MAPK, inhibition of adenylate cyclase

Several P2Y receptor subtypes will be of special interest in the present study and are discussed below:

P2 Yi receptor The P2Yi receptor is a receptor activated by adenine nucleotides (ADP and ATP), but not by uridine nucleotides (uridine 5’-diphosphate (UDP) and UTP) (von Kügelgen and Wetter 2000). ADP is the most potent naturally occurring agonist, and whether ATP is a full, partial or inactive agonist remains debated and might be species- dependent. It is the only subtype for which significant progress has been made for high- affinity and selective synthetic agonists and antagonists, e.g. 2-methylthioADP (2- MeSADP) is a highly potent agonist at the P2Y% receptor and MRS2179 is a non- selective antagonist (King et al. 2000).

The P2Y1 receptor, which upon agonist binding mainly activates PLC through a Gq/ii-protein, is distributed widely throughout the body. The P2Y% receptor was first cloned from chick (Webb et al. 1993), and then successively from turkey (Filtz et al. 1994), rat and mouse (Tokuyama et al. 1995), bovine (Henderson et al. 1995) and human tissues (Leon et al. 1996). It has been described in vascular, connective, immune and neural tissues. P2Y% receptors are thought to be mitogenic in endothelial cells

26 (Bumstock 2002a). A major functional role is the induction of platelet aggregation. Activation of the P2Yi receptor on platelets leads to platelet shape change, aggregation and [Ca^^Ji rise. ADP, secreted from platelet dense granules, can also potentiate the aggregation response induced by other agents. Targeted disruption of the P2Y% receptor has been shown to result in impaired platelet aggregation and increased resistance to thrombosis (Fahre et al. 1999; Léon et al. 1999).

P2 Y2 receptor

The P2Y% receptor, formerly called ‘P 2U receptor’, is activated by ATP and UTP with approximately equal potency (King et al. 2000) and is insensitive to nucleoside diphosphates (von Kügelgen and Wetter 2000). The receptor is widely distributed in the body. Signal transduction mechanisms are mediated by both Gi/o and Gq/n proteins. The downstream signalling events seem to depend on the cell type involved, e.g. in airway epithelial cells, secondary to PLC activation and [Ca^'*']i mobilisation, Ca^'^-sensitive Cl' channels open up, driving fluid secretion (Cressman et al. 1999). This could be of pathophysiological significance in patients with cystic fibrosis, a condition characterised by a failure to secrete Cl' ions into the airways. Treatment of cystic fibrosis with the main P2 Y2 agonist, UTP, is being pharmacologically explored (Stutts et al. 1992;

Cressman et al. 1999). P 2 Y2 receptor mRNA has been localised in human epidermal basal cells via in situ hybridisation (Dixon et al. 1999). Functional P 2 Y2 receptors have also been characterised on human ovarian cancer cells (Schultze-Mogasu et al. 2000), colorectal carcinoma cells (Hopfner et al. 1998), prostate carcinoma cells (Janssens and Boeynaems 2001), endometrial cancer cell lines (Katzur et al. 1999), and MCF-7 breast cancer cells (Wagstaff et al. 2000).

P2 Y4 receptor

Earlier studies on the P 2 Y4 receptor suggested that this receptor is highly selective for UTP over ATP. This, however, is not tme for the cloned rat P 2 Y4 receptor, whereas the human P2 Y4 receptor shows a strong preference for UTP over ATP (von Kügelgen and Wetter 2000). Diphosphate nucleotides UDP and ADP are inactive in

P2 Y4 receptors. P 2Y4 seems to have a restricted distribution to the placenta, but mRNA has also been detected in vascular smooth muscle (Harper et al. 1998).

Sources and fate of nucleotides

Nucleotides are released into the extracellular fluid from a variety of sources. ATP cannot cross plasma membranes due to its size and high charge density. Most of

27 the research into potential mechanisms has focused on release of ATP itself, but ADP and UTP release have also been reported (Saiag et al. 1995; Lazarowski et al. 1997; Homolya et al. 2000). ATP is actively released from nerves (Bumstock et al. 1970; Zimmermann 1994), glial cells (Cotrina et al. 1998), epithelial cells (Dezaki et al. 2000), endothelial cells (Milner et al. 1990; Bumstock 1999a), smooth muscle cells (Katsuragi et al. 1991), and from immune cells (Filippini et al. 1990). A number of cell types release ATP upon mechanical stimulation, including tumour cells (Pedersen et al. 1999), platelets and red blood cells (Alkhamis et al. 1990), osteoblasts (Bowler et al. 1998) and chondrocytes (Lloyd et al. 1999). Different mechanical stimuli also initiate ATP release, e.g. shear stress on endothelial cells (Milner et al. 1990; Bodin et al. 1991) and both change in cell volume (Dezaki et al. 2000) and cell stretch (Bumstock 1999a) in epithelial cells.

Sources of ATP Intracellular ATP can be released passively from dying, necrotic cells undergoing cytolysis; or it can be actively release from living cells by vesicular release or via ATP-binding cassette (ABC) proteins.

Cytolysis ATP release through cytolysis takes place after cell damage or cell death following physical or biological trauma, and can thus contribute to pathophysiological conditions. The intracellular concentration of ATP is about 2-5 mM, and damage to the cell membrane can therefore release high quantities of ATP locally (Chow et al. 1997). During trauma, nucleotides have been shown to attain levels as high as 20|iM (Bom and Kratzer^ 1984), a concentration sufficient to activate all P2 receptor subtypes. In addition, activated platelets and leukocytes release ADP and ATP at sites of tissue injury and inflammation. Apoptosis in contrast to necrosis is not a likely route for passive ATP release from the cell. Apoptotic cells compartmentalise into cell fragments (apoptotic bodies), but the cytoplasm of these compartments is still separated from the extemal milieu by an intact cell membrane. P2X? receptors have a role in the induction of cell death by both apoptosis and necrosis (Ferrari et al. 1999). The stimulation of P2X? receptors may cause further ATP release after necrotic cell lysis, but not after induction of apoptosis.

Vesicular release Vesicular release is the way ATP is released from nerve terminals, and possibly from some non-neuronal cells. In nerve terminals, ATP and other transmitters are stored

28 in pre-synaptic vesicles and released through exocytosis during neurotransmission. Recent studies suggest that vascular endothelial cells, when subjected to shear stress, release ATP by vesicular transport (Bodin and Bumstock 2001a).

ABC proteins ABC proteins have only recently been implicated in the release of ATP. They constitute a highly conserved family of ATP-dependent membrane transporters and consist of two cytoplasmic catalytic ATP-binding domains and two hydrophobic transmembrane domains, which form the translocation pathway for substrates such as amino acids, inorganic ions, sugars and proteins. The most common ABC proteins are the cystic fibrosis conductance regulator (CFTR), the sulfonylurea receptor and P- glycoprotein, product of the multidrug resistance protein gene. Although the concept that ABC proteins can also transport ATP itself across the membrane is still controversial, some studies, using specific blockers of ABC protein channels, suggest their involvement on different types of cells; e.g. ATP release by osteoblasts or Ehrlich ascites tumour cells can be inhibited by the addition of glibenclamide, a non-specific CFTR-blocker (Bowler et al. 1998; Pedersen et al. 1999). For a recent review on ATP release, see Bodin and Bumstock (2001b).

The purinergic cascade Once released, ATP is degraded to ADP, AMP and adenosine by a family of approximately 11 ectonucleotidases (ectonucleoside triphosphate diphosphohydrolases - E-NTPases) (Zimmermann and Braun 1999; Zimmermann 1999). They are located on the cell surface and form an enzyme cascade that sequentially degrades nucleoside 5’- triphosphates to their respective nucleoside 5’-di and -monophosphates, nucleosides and free phosphates or pyrophosphate, which can all appear in the extracellular fluid at the same time. This limits the extracellular actions of ATP at receptors. The breakdown products - ADP, AMP and adenosine are themselves pharmacologically active and form a purinergic cascade (Williams and Jarvis 2000). These breakdown products of ATP can have opposing effects to ATP, for example ATP can antagonise ADP actions on P 2Yi2 receptor mediated platelet aggregation, while the sedative effects of adenosine in the CNS contrast with the excitatory actions of ATP on nerve cells (Bumstock and Williams 2000). So if PI receptors are present on neighbouring cells, adenosine can initiate additional receptor-mediated functions, as well as ATP associated actions on P2 receptors. To complicate matters, these enzymes have been shown to represent possible targets of P2 receptor antagonists, e.g. suramin is an ATPase inhibitor: thus care must

29 be taken in P2 receptor antagonist studies using suramin. While UTP and UDP are active at P2 Y2, P2 Y4 and P2Yô receptors (Communi and Boeynaems 1997), evidence for the physiological role of uracil has been limited. For reviews on extracellular ATP metabolism, see Zimmermann and Braun (1999) and Zimmermann (2000).

Co-existence of A TP-consuming and ATP-generating pathways Along with the commonly accepted view of the extracellular nucleotide degradation cascade as a purine inactivating mechanism relevant for the function of purinergic receptors, an opposite ATP-generating pathway via phosphotransfer reactions has only been recently appreciated (Yegutkin et al. 2001). Recent work has demonstrated the co-existence of two opposite ectoenzymatic pathways on the cell surface, where constitutive ATP release, continuous re-synthesis of high energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent nucleoside inactivation (Yegutkin et al. 2002). These data demonstrate the co-existence of opposite, ATP-consuming and ATP- generating, pathways on the cell surface, and provide a novel mechanism for regulating the duration and magnitude of purinergic signalling and for cells maintaining specific local concentrations of extracellular nucleotides.

30 Physiological roles of ATP

Purinergic signalling plays important roles in nervous and non-nervous tissues, including both short-term, fast actions (neurotransmission and secretion), and long-term, slower, trophic actions (development and regeneration) (Bumstock 1997).

Short-term purinergic signalling

ATP as a co-transmitter in the nervous system ATP acts as a co-transmitter in many nerves of both peripheral and central nervous system. Sympathetic co-transmission of noradrenaline and ATP has been demonstrated in smooth muscle of the vas deferens and in blood vessels. Similarly, acetylcholine and ATP are co-transmitters in various peripheral and central synaptic terminals, including parasympathetic nerves supplying the urinary bladder and motor nerves of skeletal muscle. Sensory motor nerves can co-release ATP and calcitonin gene-related peptide and substance P. Neurones located in the myenteric plexus co release ATP, NO and VIP, with the proportions varying in different regions of the gut. In the CNS, there is evidence that ATP is co-released with glutamate. For a recent review on purinergic co-transmission, see Bumstock (1999b). Exocrine and endocrine secretion Another short-term signalling mechanism of ATP is the regulation of ion- transport in epithelial cells. ATP and UTP stimulate chloride ion transport and these actions are important in airways epithelia, where the net effect is to add liquid to airway surfaces. Patients with cystic fibrosis have a disorder where cells are relatively impermeable to chloride ions, resulting in viscous secretions that are high in salt and low in water, leading decreased mucociliary clearance and chronic airway infection. Trials of aerosol application of UTP in cystic fibrosis patients have shown improved mucociliary clearance without affecting airway calibre (Abbracchio and Bumstock 1998). ATP also regulates gastric acid secretion and secretions from parotid and lacrimal acinar glands. ATP also has important endocrine actions, e.g. in the stimulation of insulin secretion by pancreatic |3-cells (see Abbracchio and Bumstock (1998) for a review). P2 receptors have also been found on pituitary cells, adrenal medulla chromaffin cells, PC 12 phaeochromocytoma and neuroblastoma cells, thyroid cells, luteal and granulosa cells of the ovary and Sertoli cells (Abbracchio and Bumstock 1998). Although the

31 exact role of these receptors in the function of these cells is still unknown, these findings suggest that ATP may have a role in the regulation of endocrine function at some level. Regulation of vascular tone In the vascular system, short-term purinergic signalling events associated with the control of vascular tone by ATP released from nerves and endothelial cells have been clearly demonstrated (Bumstock 1990; Ralevic and Bumstock 1991; Bumstock and Ralevic 1994; Bumstock 1999a). Regulation of immune cell function

Both PI (A 2a) and P2X?, P2Yi and P2Y2 receptors are expressed in immune cells. Activation of specific receptor subtypes on these cells may result in the modulation of cytokine release, natural killer cell activity and induction of specialised functions such as migration, secretion, phagocytosis and cell death (Abbracchio and Bumstock 1998; Bumstock 2001a).

Long-term purinergic signalling

There is increasing evidence that ATP, signalling through PI and P2 receptors, plays a major role in embryonic development and cell growth, proliferation and differentiation, acting both as a positive and negative regulator. Cell proliferation and growth Nucleotides can both stimulate cell cycle progression and inhibit cell growth, depending on their concentration, the physiological state of target cells and receptor expression (Harada et al. 2000). Stimulation of DNA synthesis and cell proliferation by extracellular nucleotides has been demonstrated in Swiss mouse 3T3 and 3T6 fibroblasts, thymocytes, haematopoietic cells, smooth muscle cells, endothelial cells, primary astrocytes and astrocytoma cell lines (Abbracchio and Bumstock 1998; Bumstock 2002a). In 3T3 and A431 cells, these effects seem to be mostly regulated by low ATP concentrations (micromolar range) acting via P2Y receptors linked to stimulation of PLC and subsequent [Ca^^ji increase (Huang et al. 1989). The same authors have shown that ATP can also act synergistically with various growth factors to cause mitogenic effects. A recent review has investigated the evidence that P2Y receptor-activated mitogenesis could act via mitogen-activated protein kinase (MAPK)- dependent pathways (Bumstock 2002a). ATP and UTP activate the 42-kDa isoform of MAPK and

32 this activation is regulated by PKC in endothelial cells (Graham et al. 1996). Regulation of rat brain capillary endothelial cells via P2Y receptors (probably P 2 Y2 and/or P 2Y4 receptors since UTP was equipotent with ATP) has been shown to be coupled to Ca^"^, phospholipase C (PLC) and MAPK (Albert et al. 1997). The modulation of cell growth and proliferation may play crucial roles following trauma, stress and hypoxia. At sites of damage, nucleotide concentrations are massively increased and may thus contribute to the initiation of healing mechanisms, ultimately leading to tissue repair and regeneration.

Interactions of purines and pyrimidines with growth factors Purines may modulate the activities of growth factors at their target systems. Synergistic interactions between extracellular ATP and polypeptide growth factors lead to greatly enhanced mitogenesis in primary astrocytes (Neary et al. 1994) and neuroblastoma cells (Wang et al. 1990). In PC 12 cell cultures, neurite extension by guanosine, GTP and AlT-082 was synergistic with that of NGF (Gysbers and Rathbone 1996). In 3T3 and A431 cells, both EGF and ATP had very little mitogenic effects when tested alone, but synergistically stimulated DNA synthesis when added to cells together (Huang et al. 1989). ATP, when added to NA and neuropeptide Y stimulated proliferation of human vascular smooth muscle cells (Erlinge et al. 1993). Purines and pyrimidines can not only act as novel growth factors by inducing trophic actions at target cells, but can also interact with growth factors to cause synergistic effects.

Purinergic signalling in embryonic development ATP and adenosine can play important roles from the beginning of life, e.g. some studies suggest that ATP serves as a key sperm-to-egg signal in the process of fertilisation (Foresta et al. 1992). Developmentally regulated expression of P2 receptors has been demonstrated in a variety of tissues including chick muscle, retina and gastrointestinal tract, pointing to a critical role for P2 receptors in maturation and acquisition of highly specialised functions. As an example, our lab has shown that in developing rat skeletal muscle, timing of P 2X2 receptor expression was closely related to the re-distribution of acetylcholine receptors to the motor end-plate (Ryten et al.

2 0 0 1 ). In many cases, once specialised tissue functions have been obtained, the receptors are not required anymore and likely to be desensitised. However, receptors and key signals might be expressed again, if necessary, under specific pathophysiological conditions, such as regeneration following trauma and injury. As an

33 example, chick muscle regains its previously lost response to ATP after denervation followed by reinnervation (Wells et al. 1995). Other studies suggest a role for P2Y receptors in the early formation of the nervous system (Bogdanov et al. 1997).

Purinergic signalling in apoptosis and cancer Induction of apoptosis seems to play an important role in ATP anti-cancer activity. Apoptosis is a normal and deliberate process that takes place when cell death is part of an organised tissue reaction, such as can be found in embryogenesis, tissue atrophy, aging and in some cases tumour regression. In normal healthy adult tissue, apoptosis can be regarded as a physiological means to complement mitosis in maintaining tissue size, and indeed failure to undergo apoptosis has been implicated in tumour development (King and Cidlowski 1995). As opposed to necrosis, apoptosis progresses through a series of well-defined morphological and biochemical stages occurring in the nucleus as well as in the cytoplasm of the dying cell. This includes condensation and margination of chromatin towards the inner nuclear membrane, followed by nuclear disruption into apoptotic bodies and chromatin cleavage into high molecular weight DNA fragments, which are subsequently further degraded (Chow et al. 1997). Apoptosis is induced by glucocorticoids, lymphotoxins, tumour necrosis factor and related cytotoxins (King and Cidlowski 1995) and by ATP (Di Virgilio 1995). P 2X7 receptor activation by ATP triggers apoptosis to facilitate removal of cancerous or infected cells from tissues (Bumstock and Williams 2000). The P2X? receptor is unlike other P2X receptors because it is a bifunctional molecule, that can be triggered to act as a channel permeable to small cations, or on prolonged stimulation form a cytolytic pore permeable to large hydrophilic molecules up to 900 Da (Surprenant et al. 1996). The opening of this pore results in the increase in intracellular cytosolic free calcium ions. The P2X? receptor is involved in the release of IL-ip (Ferrari et al. 1997a) (presumably mediated via the activation of IL-ip-converting enzyme (ICE), which is now known to be part of the caspase family) and the induction of cell death (Zheng et al. 1991; Ferrari et al. 1996). Adenosine also has cytotoxic effects, although the mechanism remains unclear. It is likely that purinoceptors are not involved because the toxicity of adenosine can be prevented by blockers of adenosine uptake, suggesting a possible intracellular site of action of adenosine (Chow et al. 1997). ATP and adenosine have been shown to have a variety of effects on cancers: improvement of cancer cachexia, inhibition of tumour growth and a synergistic action

34 on tumours when administered with chemotherapeutic drugs. These will be discussed below.

Inhibition of tumour growth The anti-cancer activity of adenine nucleotides was first demonstrated by Rapaport (Rapaport 1983). Extracellular levels of ATP below lOOpM (Rapaport 1983; Weisman et al. 1988; Abraham et al. 1996), between 100-500pM (Mure et al. 1992; Fang et al. 1992; Spungin and Friedberg 1993; Vandewalle et al. 1994), between 500- lOOOpM (Zheng et al. 1991; Spungin and Friedberg 1993) and above lOOOpM (Zheng et al. 1991; Hatta et al. 1993, 1994), exerted cytostatic and cytotoxic effects on many transformed cell lines. In general these effects appeared greater than the effects on non transformed cells. In animal cell lines, ATP inhibited the growth of transformed fibroblasts (Weisman et al. 1988; Kitagawa et al. 1988; Belzer and Friedberg 1989), leukaemia cells (Chahwala and Cantley 1984; Hatta et al. 1993, 1994), mastocytoma cells (Filippini et al. 1990), lymphoma cells (Di Virgilio et al. 1989), thymocytes (Zheng et al. 1991), and melanoma cells (Mure et al. 1992). In human cell lines ATP inhibited the growth of pancreatic carcinoma cells, colon adenocarcinoma cells (Rapaport 1983, 1988), melanoma cells (Rapaport 1983; Kitagawa et al. 1988), androgen-independent prostate carcinoma cells (Fang et al. 1992), and breast cancer cells (Rapaport 1983; Spungin and Friedberg 1993; Vandewalle et al. 1994; Abraham et al. 1996). Similar exposure of non-transformed cell lines to ATP produced relatively less inhibition of cellular growth compared with transformed cells or no inhibition at all (Rapaport 1983; Mure et al. 1992; Spungin and Friedberg 1993; Hatta et al. 1993, 1994). In mice with CT26 colon tumours, daily intraperitoneal injection of ATP (25-50 mM) significantly inhibited tumour growth (Rapaport 1988; Rapaport and Fontaine 1989a, 1989b; Rapaport 1990). ATP induced growth inhibition was also demonstrated in mice with lymphomas (Nayak et al. 1990), fibrosarcomas (Froio et al. 1995), Ehrlich ascites tumours (Pal et al. 1991; Lasso de la Vega et al. 1994) and breast tumours (Abraham et al. 1996). Furthermore, intraperitoneal ATP administration resulted in a significantly prolonged survival of these animals (Lasso de la Vega et al. 1994; Estrela et al. 1995; Obrador et al. 1997). Inhibition of tumour growth was also demonstrated in athymic nude mice, suggesting that the anti-cancer activities of extracellular ATP was not likely to be due to an ATP-mediated enhancement of host immunological functions, but rather due to the direct action of ATP on tumour tissue (Rapaport 1988).

35 In an open-labelled phase U study, 15 untreated patients with advanced non small cell lung cancer (stage UTB/TV) received 1-4 intravenous ATP courses (50-65 pg/kg/min for 96 hours) administered at 4 week intervals. No objective complete or partial tumour responses to ATP were reported, although stable disease was found in about two thirds of the patients during the courses of treatment (Haskell et al. 1998). The major toxic side effects were chest pain and dyspnea, leading to the cessation of treatment in 5 patients.

Cancer cachexia Cancer cachexia is a syndrome of progressive weight loss associated with depletion of liver and skeletal muscle energy stores. This depletion is caused by elevated lipolysis, protein breakdown and gluconeogenesis (Agteresch et al. 1999). Dietary and enteral supplements fail to reverse the cachexia. In liver tissue (Schneeberger et al. 1989; Tsuburaya et al. 1995) and in skeletal muscle (Schneeberger et al. 1989) of tumour bearing rats, significantly lower ATP levels have been demonstrated and this is associated with increased gluconeogenesis (Tsuburaya et al. 1995). Daily intraperitoneal injections of 25mM ATP, AMP or adenosine for 10 consecutive days into mice with colon tumours induced a significant inhibition of host weight loss (Rapaport and Fontaine 1989a, 1989b). This inhibition was associated with expansion of hepatic ATP pools (Rapaport and Fontaine 1989a). Rapaport suggested that ATP may inhibit Cori cycle activity (gluconeogenesis from lactate followed by reconversion of glucose to lactate in peripheral tissues), which is a potential means of inhibiting weight loss (Rapaport 1990). In a non-randomised clinical trial, intravenous infusion of ATP Tor 96 hours at 28 day intervals at doses of 50pg/kg/min to patients with advanced non-small cell lung cancer increased ATP pools in red blood cells, inhibited weight loss, reduced cachexia and improved survival (Rapaport 1997). In a subsequent randomised trial (Agteresch et al. 2000a) in patients with advanced (Stage IQB or IV) non-small-cell lung cancer, intravenous ATP reduced weight loss and reversed decreases in serum albumin, elbow flexor muscle strength and quality of life measures. Positive effects on body weight, muscle strength and albumin concentration were especially marked in cachectic patients. No change in body composition over the 28-week follow-up period was found in the ATP group, whereas, per 4 weeks, the control group lost 0.6 kg of fat mass, 1.8% of arm muscle area and 0.6% of body cell mass/kg body weight and decreased 568 KJ/day in energy intake. Appetite remained stable in the ATP group but decreased

36 significantly in the control group (Agteresch et al. 2002). Most ATP courses in this group of cancer patients were without side effects (64%), and side effects occurring in the remaining courses were mild and transient, resolving within minutes after decreasing the infusion rate (Agteresch et al. 2000b).

Synergistic action of ATP with chemotherapeutic agents In cancer cell lines, ATP may enhance the efficacy of several chemotherapeutic agents. Addition of 200-5OOpM ATP to doxorubicin in cultures of human ovarian carcinoma cells doubled the cell mortality, when compared with doxorubicin alone. 30- 50% more doxorubicin accumulated in the cancer cells when given together with ATP, whereas in healthy human fibroblasts, practically no effect of ATP on doxorubicin uptake was observed (Maymon et al. 1994). In a mouse melanoma cell line (clone-M3), 500|ig/kg/min ATP administration markedly increased the passive permeability for chemotherapeutic agents such as fluorouracil, doxorubicin, mitomycin and nimustine. The cytotoxic effects of these chemotherapeutic agents were additively potentiated by treatment with ATP. Vincristine combined with ATP showed even a synergistic cytotoxic effect; the effective concentration of vincristine was lowered 10-50 fold by ATP treatment (Mure et al. 1992). The effect of potentiating chemotherapeutic agents was demonstrated in vivo after administration of adenosine. In mice inoculated with B-16 melanoma cells, adenosine (5mmol/L) was injected 5 days before administration of cyclophosphamide (50mg/kg). This combined treatment reduced the number of melanoma foci by 60%, while the chemotherapy alone only reduced them by 45%. Moreover, a protective effect of adenosine against chemotherapy-induced decrease of leukocyte counts was seen in this study (Fishman and Bar-Yehuda 1998).

Radiotherapy In various animals, the protective effects of intramuscular or intraperitoneal ATP injections against radiation damage enhanced survival rates from 5 to 50% (Nikolov et al. 1986), 4 to 40% (Tikhomirova et al. 1984), 40 to 85% (Szeinfeld and de Villiers

1992a) and from 26 to 86 % (Szeinfeld and de Villiers 1992b). In addition intravenous

ATP-MgCl 2 infusion (60pmol/L/kg) in pigs offered significant cytoprotection from pelvic radiotherapy (Senagore et al. 1992). ATP infusion led to diminished colorectal seromuscular ischaemia, and reduced skin and subcutaneous injury (Senagore et al. 1992). When radiation was given in combination with ATP, the frequency of formation of aberrant mitoses in epithelial cells of the mouse cornea was lower than when no ATP

37 was given (Vorozhtsova et al. 1987). This combination reduced the tumour growth rate of transplanted fibrosarcomas in mice (Froio et al. 1995) and led to significant regression of Ehrlich ascites tumours in mice (Estrela et al. 1995).

Adverse effects In general, ATP and adenosine induce similar adverse effects when given as either continuous intravenous infusions or by intravenous bolus injection. Adverse effects of ATP and adenosine include: general discomfort (Ferreira et al. 1996), breathing deeper or more frequently (Davies et al. 1951; Perrot et al. 1984; Watt et al. 1986; Biaggioni et al. 1987; Jonzon et al. 1989; DiMarco et al. 1990), headache (Jonzon et al. 1989; DiMarco et al. 1990; Verani et al. 1990), flushing, chest pressure or chest pain and nausea (Perrot et al. 1984; Sylven et al. 1986; Rankin et al. 1989, 1990; Verani et al. 1990; DiMarco et al. 1990; Sethi et al. 1994; Domanovits et al. 1994). Sinus bradycardia (Rankin et al. 1989; DiMarco et al. 1990) and atrial fibrillation (Belhassen et al. 1984; Strickberger et al. 1997) have also been observed in patients receiving bolus injections of adenosine. The adverse effects of bolus administration of ATP and adenosine are generally mild and transient because of the short plasma half-life (0.6 to 1.5 seconds) (Moser et al. 1989). During continuous intravenous infusion of ATP in a phase I study in patients with advanced cancer, the adverse effects observed included a feeling of chest tightness without frank pain and a sensation of needing to take a deep breath (Haskell et al. 1996). No significant haematological toxicity was observed. The most appropriate dosage of ATP in patients with advanced cancer was 50pg/kg/min. Adverse > effects during intravenous infusion resolved within seconds after discontinuing the ATP infusion. Intravenous adenosine infusion could induce transient haemodynamic effects, including increased heart rate, small variations in systolic blood pressure and a small but significant decrease in diastolic pressure and mean arterial pressure in conscious patients and volunteers (Faulds et al. 1991).

38 Summary of features of purinergic signalling

Purinergic signalling can occur in five different ways: from neurons to neurons (e.g. in the central nervous system and in peripheral ganglia); from nerves to peripheral tissues (e.g. in smooth muscle contraction and in secretion of epithelial cells); from peripheral tissues to nerves (e.g. in the mediation of pain); autocrine (e.g. in controlling secretion); and paracrine (e.g. ATP alone or together with either other transmitters, nitric oxide or growth factors induces apoptosis, secretion, proliferation, immune cell activation, and regulation of vascular tone). Thus extracellular ATP serves generally for communication between cells. Mechanical stimuli are integrated into biochemical responses (e.g. ATP release), which then initiate coordinated responses of respective tissues (e.g. secretion, contraction, proliferation or cell death). ATP often works synergistically with other transmitters (e.g. in co-transmission in nerves and in the induction of proliferation together with growth factors).

39 IL SKIN

The skin is the largest organ of the human body, exceeding 1.7m^ on average and accounting for almost one sixth of the total body weight. It comprises a sheet-like investment of the entire body that adapts to its contours and conforms to the movements of the organism within. It acts as a physical barrier to prevent desiccation from evaporation, is able to withstand mechanical, chemical and microbial insults and has important thermoregulatory and sensory roles. Skin is composed of two distinct layers, the superficial epidermis and a deeper dermis. This thesis will focus on events within the epidermis and so a summary of areas of skin biology that are the focus of the results chapters is presented here.

The epidermis

The epidermis is a thin layer of stratified squamous epithelium that rests on top of a basement membrane, which separates it and its appendages, including the hair follicles and sweat glands from the underlying mesenchymally derived dermis (Fig 1.3). The epidermis is a unique structure which permits us to study the processes of cell growth and renewal as well as cellular differentiation. Epidermal architecture The epidermis is derived from the embryological ectoderm and its principle cell type is the keratinocyte. Other cell types occur in the epidermis in relatively small numbers. These are Langerhans cells, Merkel cells, melanocytes and dendritic cells. While these cells are functionally important, they are only minor epidermal components and do not greatly influence epidermal architecture. This thesis will focus on the biology of epidermal kératinocytes. Kératinocytes are organised into distinct cell layers, which form four morphologically and spatially distinct regions (Fig 1.3). The stratum germinativum or basal layer, containing columnar cells, is the deepest layer and the cells are in contact with an underlying basement membrane. The stratum spinosum, or spinous layer, contains several layers of polyhedral cells. The stratum granulosum or granular layer, contains flattened, nucleated cells that have distinctive cytoplasmic keratohyalin granules. The stratum comeum or comified layer, contains thin, flat, anucleate cells that are the end product of terminal differentiation. This is the most superficial layer of the epidermis.

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Figure 1.3: The skin and its appendages. Human skin consists of the epidermis and dermis, separated by a basement membrane. The epidermis is a stratified squamous epithelium that is composed of several cell layers (seen in close-up, right). Resting on the basement membrane is the basal layer (BL), consisting of stem cells and proliferating transit-amplifying cells. The basal layer stratifies to give rise to differentiated cells layers of the spinous layer (SL), granular layer (GL) and the stratum corneum (SC). Also shown is a cross section of a hair follicle, which consists of an outer root sheath that is contiguous with the basal epidermal layer. At the bottom of the follicle is the hair bulb, made from proliferating matrix cells. The transit-amplifying matrix cells terminally differentiate to generate the different cell types of the follicle. Also shown is the bulge, which is part of the outer root sheath and is where epidermal stem cells reside. The dermal component of the hair follicle is the dermal papilla, which consists of specialised mesenchymal cells surrounded by the hair matrix cells. Taken from Fuchs and Raghavan (2002).

41 Proliferation takes place in the basal layer of the epidermis. Kératinocytes that leave this layer stop dividing and undergo terminal differentiation as they move through the suprabasal layers. Cells in the stratum comeum are exquisitely specialised to form a protective barrier between the body and the environment, due to the assembly of an insoluble layer of transglutaminase-crosslinked protein, the comified envelope, undemeath the plasma membrane (Watt 2002). Comified cells are continuously shed from the surface of the epidermis and therefore new differentiated cells must be produced throughout life. Replenishment of the differentiated compartment of the epidermis depends upon the proliferation of a subpopulation of cells in the basal layer known as stem cells. Keratinocyte proliferation and stem cells A single adult stem cell has sufficient proliferative capacity to produce enough new epidermis to cover the body surface (Rochat et al. 1994; Watt and Hogan 2000). Stem cells can be defined as cells that have an unlimited capacity for self-renewal and the ability to generate daughter cells that undergo terminal differentiation (Watt 2002). In human skin, there are two subsets of stem cells - those found in the basal layer of the interfollicular epidermis and others in the bulge region of hair follicles (Fig 1.3). Stem cells in the bulge region of hair follicles of furry mammals are multipotent and have the capacity to regenerate epidermis as well as hair follicles and sebaceous glands. Whether basal epidermal stem cells can generate hair follicles remains unclear (Fuchs and Raghavan 2002). Most cells within the basal layer are the rapidly dividing progeny of stem cells (Jones and Watt 1993; Jones et al. 1995). These are the transit-amplifying cells. They undergo a limited number of divisions before they withdraw from the cell cycle, commit to terminal differentiation, detach from the basement membrane and begin their journey towards the surface of the skin (Barrandon and Green 1987a; Watt 1998). Cells that reach the body surface are dead, enucleated, flattened cells (squames) that are subsequently sloughed and are continuously replaced by inner cells that move outwards. So the epidermis is in a constant state of dynamic equilibrium, completely renewing itself every 2 weeks throughout 1 life (Eckert et al. 1997). Stem cells have been studied in vitro and have the following characteristics: the selective down-regulation of certain proteins, e.g. the transferrin receptor (Tani et al. 2000), 14-3-3a (a nuclear export protein) and the expression of cytokeratins K19 and K15. Human epidermal stem cells express higher levels of pi integrins than transit- amplifying cells and antibodies to pi integrins can be used to isolate viable keratinocyte

42 populations enriched for stem cells directly from the epidermis (Jones and Watt 1993; Jones et al. 1995). Staining sections of human epidermis with anti-pl integrin antibodies suggests that stem cells are clustered, rather than distributed as single cells (Jones et al. 1995). Most of the markers that are expressed in epidermal stem cells are also expressed in the transit-amplifying offspring, which raises the question of whether these markers function to maintain stem cell character or proliferative capacity. An example of this is p63, a p53 homologue that is expressed throughout the basal layer of the epidermis (Pellegrini et al. 2001). The p63 protein has been implicated in repressing the transcription of the epidermal growth factor receptor (EGFR) gene and the genes regulated by p53 and the cell cycle (Yang et al. 1999), and any of these functions could maintain stem cells in a slow cell-cycling state. Mice that lack Trp63, which encodes p63 have a very thin epidermis and severe defects in epidermal proliferation, stratification and differentiation (Yang et al. 1999; Mills et al. 1999). The early onset of these defects, together with the normal expression of Trp63 and its null phenotype, indicates a role for p63 in maintaining transit-amplifying epidermal cell character rather than that of stem cell.

Regulation of stem cell fate pi integrins, p-catenin and c-Myc can all regulate epidermal stem cell fate autonomously (Watt 2002). pi integrin pi integrin levels differ between stem and transit-amplifying cells and integrins are known to regulate the onset of terminal differentiation (Watt 2002). To show the effect of removing pi integrin, a dominant negative integrin was generated by expressing a chimera of the extracellular domain of CDS and the cytoplasmic domain of the piA integrin subunit (Zhu et al. 1999). The expression of CDSpi reduced surface pi levels, adhesiveness and MAPK activation in response to pi integrin ligation. Exit from the stem cell compartment was stimulated and the proportion of clones attributable to the transit-amplifying population increased. Adhesiveness and proliferative potential were restored by overexpressing the wild type chick pi subunit in cells that expressed CDSpi. Constitutive MAPK activation did not affect the adhesiveness or proportion of stem cell clones in control kératinocytes. So, from these experiments it was concluded that pi integrins and MAPK co-operate to maintain the epidermal stem cell compartment in vitro (Zhu et al. 1999).

43 fi-catenm Stem cell enriched cultures of human kératinocytes have a higher level of free, non-cadherin associated P-catenin than transit-amplifying cells (Zhu and Watt 1999). Expression of stabilised, N-terminally truncated p-catenin increased the proportion of stem cells to almost 90% of the proliferative population in vitro (Fig 1.4a). A dominant negative P-catenin mutation also stimulated the exit of kératinocytes from the stem cell compartment (Zhu and Watt 1999).

a stem cells stem cells c-Myc ^ c-Myc i Transitsit Icells Transit cells 1 i Interfollicular Interfollicular eoidermis eoidermis

Figure 1.4: Schematic interpretation of the effects of c-Myc and p-catenin on the epidermal stem cell compartment in culture. Taken from Watt (2002).

c-Myc Another regulator of stem cell fate is the proto-oncogene c-Myc (Gandarillas and Watt 1997). In contrast to the effect of over-expressing p-catenin, activation of c- Myc stimulated exit from the stem cell compartment (Fig 1.4b). Myc is thought to be a downstream target of P-catenin, suggesting that an autoregulatory loop may exist in the epidermis (Zhu and Watt 1999). c-Myc is expressed in several subpopulations of epidermal cells, including the basal layer of the interfollicular epidermis and the bulge of the hair follicle, which is a reservoir of stem cells (Bull et al. 2001). When c-Myc transgenes are over-expressed in mouse epidermis via the keratin 14 promoter, adult mice lose their hair and develop spontaneous open wounds (Waikel et al. 2001; Arnold and Watt 2001). Proliferation is stimulated, but interfollicular kératinocytes still undergo normal terminal differentiation (Arnold and Watt 2001). The number of BrdU label retaining cells is reduced (Waikel et al. 2001), although whether this represents true depletion of the stem cell compartment remains to be investigated.

44 Keratinocyte differentiation Growth and differentiation are tightly linked processes in the epidermis, which need to be precisely balanced. If there are too many dividing cells, hyperproliferative disorders of the skin result, such as psoriasis and basal or squamous cell carcinomas. Conversely premature differentiation can yield a thin epidermis, as seen in sun-damaged or aged skin. The epidermis controls this balance in part by orchestrating a transcriptional programme that produces temporally and spatially distinct epidermal cells, each able to carry out tasks that are specific to their position in the skin (Fuchs and Raghavan 2002). As kératinocytes withdraw from the basal layer, they stop dividing and induce a programme of terminal differentiation that will ultimately allow them to function as the barrier for the skin.

The skin: a protective barrier Basal layer kératinocytes are relatively undifferentiated cells that are anchored to the basal lamina by hemidesmosomal junctions. As previously described, it is this layer that provides a continuous supply of new cells to repopulate the epidermis (Watt 1998). Cells of the stratum spinosum are found immediately above the basal layer and are in the main incapable of division - i.e. are committed to terminal differentiation. This layer is characterised by the presence of extensive desmosomal connections between cells, which can be seen as ‘spines’ under light microscopy. Some of the early markers of keratinocyte differentiation (ie involucrin and transglutaminase) are first expressed in this layer (Holbrook and Wolff 1987). Cells in the stratum spinosum progressively mature, becoming more flattened as they move upwards through the epidermis. The stratum granulosum is distinguished by the presence of granules. These granules contain products of keratinocyte differentiation that are used to assemble various terminal keratinocyte structures, including the comeocyte membrane. These granules contain loricrin (Steven et al. 1990), filaggrin (Steven et al. 1990), cystatin-a (Takashashi et al. 1992) and lipids that are used in the assembly of the comeocyte membrane (Elias et al. 1988). In addition to these specialised organelles, granular layer cells contain all of the organelles and activities associated with intact cellular metabolic functioning, indicating that they are still viable even as they approach the final steps in differentiation. The stratum lucidum, also called the transition zone, is the layer that separates the dead from the living epidermal layers. This is a region of intense enzymatic activity in which cellular organelles and nucleic acids are destroyed by the actions of proteases.

45 nucleases and other enzymes. However, simultaneous with the destruction of cell synthetic capacity are the final steps in assembly of the keratin intermediate filament network and comified envelope. The comified cells (comeocytes) represent the terminal stage in keratinocyte differentiation and consist of a stabilised array of keratin filaments contained within a covalently cross-linked protein envelope (Matoltsy and Matoltsy 1966). These cells, which occupy the stratum comeum are dead, flattened, polyhedrons that are uniquely adapted to provide a protective surface. Disulphide bond stabilised keratin intermediate filament bundles course through their interior and connect at various points to the comified envelope. This consists of an array of proteins that are covalently linked via e- (y-glutamyl)-lysine protein-protein cross-links (Holbrook and Wolff 1987) via the action of transglutaminases (Rice et al. 1994). Billions of adjacent comeocytes are held together by modified desmosomes and by an interlocking system of ridges and grooves (Holbrook and Wolff 1987) and form the protective epidermis.

Keratins Throughout the epidermis, kératinocytes synthesize cytokeratins (keratins), a group of heterogeneous a-helical molecules which organise into 8 -lOnm tonofilaments. However, the types of keratins expressed changes with progression along the differentiation pathway. Keratin filaments form a basket network around the cell nucleus with spokes that extend through the cell cytoplasm to interact at the cell surface with desmosomes and hemidesmosomes. This cytoskeleton provides the epidermis with strength and suppleness. Keratins were first identified in the 1970s. At least 30 different keratin molecules have been described (Morley and Lane 1994). Keratins are divided into two groups: type I keratins - generally smaller and acidic (MW 40-64) and type II keratins - larger and relatively more basic (MW 53-67). Cytokeratins have been catalogued according to their charge and molecular weight (Moll et al. 1982). In this thesis, specific keratins are referred to by their Moll catalogue numbers. Keratins are normally expressed in matching pairs of type I and type II subunits, both of which are essential for the formation of tonofilaments. During normal epidermal differentiation, cytokeratins, K5 and K14 are expressed in the basal keratinocyte layer (Fuchs and Green 1980), while cytokeratins K1 and KIO are expressed in the suprabasal layers (Woodcock-Mitchell et al. 1982). In addition, in damaged skin, psoriasis, and in wound healing, cytokeratins K 6 and K16 are expressed in the epidermis. Likewise in normal epidermis, involucrin (Rice and Green 1977), loricrin (Hohl et al. 1991) and

46 filaggrin (Steven et al. 1990) and transglutaminase 1 (TGl) (Thacher and Rice 1985) are expressed in the suprabasal but not basal layers. Agents that affect keratinocyte function Kératinocytes express multiple receptors for many different factors that affect cellular function. Kératinocytes also produce cytokines that can act in an autocrine (activation of receptors on the same cell), paracrine (affecting cells in the local environment) and chemotactic (inducing the migration of cells) manner. These form a complex cascade of interacting events. These cytokines include interleukins, growth factors, colony stimulating factors and chemokines. Keratinocyte derived cytokines have a major role in inflammatory processes, immunoregulation, growth and repair, allowing epidermal kératinocytes to communicate with and influence each other as well as Langerhans cells, dermal fibroblasts, endothelial cells and infiltrating T-cells (Uchi et al. 2000). Cytokine production is stimulated by injury (including ultraviolet light) and infections (endotoxins, antigen-T cell reactions), and is reduced by corticosteroids. This is a vast area of study and some of the main cytokines and other agents that affect keratinocyte function are summarized in Table 1.3.

Table 1.3: Role of various agents in regulating keratinocyte function Compound Proliferation Differentiation

IGF-I Enhanced EGF Enhanced TGF-a Enhanced Cholera toxin Enhanced TGF-pi Reduced Enhanced KGF Enhanced IFN-y Reduced Enhanced Retinoid Reduced Vitamin D Reduced, depending on concentration Enhanced Hydrocortisone Enhanced Calcium Reduced Enhanced

IGF-1= insulin like growth factor 1; EGF= epidermal growth factor; transforming growth factor; KGF= keratinocyte growth factor; IFN=interferon.

47 Agents that enhance keratinocyte proliferation alone Insulin-like growth factor I The insulin-like growth factor signalling system consists of insulin-like growth factors (IGF-I and IGF-II), the insulin-like growth factor receptor (IGFR) and the insulin-like growth factor binding proteins (IGFBP-I,-2,-3,-4,-5,-6) (Clemmons 1992). Insulin-like growth factor I acts on cells by binding to a rra«5-membrane receptor kinase that tyrosine phosphorylates intracellular proteins (Rechler and Nissley 1990). Both IGF-I and IGF-II are important growth regulators in the epidermis and have shown to be important for normal epidermal function. Insulin-like growth factors stimulate keratinocyte migration (Ando and Jensen 1993) and proliferation (Krane et al. 1991); (Neely et al. 1991). The IGFR is localized in the basal layer of the epidermis (Krane et al. 1991). Epidermal growth factor and TGFa Epidermal growth factor (EGF) enhances both keratinocyte migration and proliferation (Barrandon and Green 1987b; Chen et al. 1993; Rheinwald and Green 1977), as does transforming growth factor-a (TGF-a) (Barrandon and Green 1987b). Epidermal growth factor has been reported to directly regulate cytokeratin K6 and K16 expression when kératinocytes are maintained in a rapidly proliferating state (Jiang et al. 1993). Both growth factors act via the same receptor. Cholera Toxin Cholera toxin is known to stimulate keratinocyte proliferation (Rheinwald and Green 1975) and to activate adenosine 3 %5'-cyclic monophosphate (cAMP)-dependent second messengers. In kératinocytes, adenosine and histamine induce adenylate cyclase and induce transient increases in intracellular calcium. Epinephrine produces a similar transient calcium increase. Dibutyryl cAMP and forskolin (a direct adenylate cyclase activator) also increase intracellular calcium. These results suggest that these agents act to stimulate keratinocyte proliferation via a cAMP-dependent pathway and that part of the response is due to a transient increase in intracellular calcium (Yasul et al. 1992). Cholera toxin and other activators of adenylate cyclase do not appear to regulate cell differentiation. Keratinocyte growth factor Keratinocyte growth factor (KGF) is a 28-kDa protein (Rubin et al. 1989). KGF is a potent epithelial mitogen that is secreted by dermal fibroblasts and acts locally on epidermal kératinocytes in a paracrine fashion (Werner and Smola 2001). KGF is a member of the fibroblast growth factor (FGF) family of growth regulatory proteins

48 (Aaronson et al. 1990). Kératinocytes rapidly release the cytokine IL-1 upon stress, like UV irradiation, surface damage, and wounding, which in turn stimulates the expression of KGF in fibroblasts (Werner 1998). This has led to the proposal of a double paracrine loop (Werner and Smola 2001), which exists between epidermal kératinocytes and dermal fibroblasts possibly to promote epidermal regeneration. Over-expression of KGF in the basal epidermal layer of transgenic mice resulted in epidermal hyperplasia and early papilloma formation, demonstrating that KGF induces proliferation but not differentiation of kératinocytes (Guo et al. 1993). These results correlated with findings that KGF reduces the ability of kératinocytes to initiate terminal differentiation and to undergo programmed cell death (Hines and Allen- Hoffman 1996). GM-CSF The expression of granulocyte-macrophage colony stimulating factor (GM-CSF) is induced in dermal fibroblasts in the presence of kératinocytes (Smola et al. 1993) and keratinocyte derived IL-1 appears to be responsible for this induction (Szabowski et al. 2000). It is possible that KGF and GM-CSF might act synergistically in the control, of keratinocyte proliferation and differentiation (Szabowski et al. 2000). Targeted expression of GM-CSF in basal kératinocytes of transgenic mice resulted in higher mitotic activity of these cells, but epidermal thickness and differentiation were normal in the skin of mice over-expressing GM-CSF. The homeostasis was in fact being maintained by increased apoptosis in the epidermis (Breuhahn et al. 2000). Therefore, GM-CSF induces both keratinocyte proliferation and apoptosis.

Proteins that regulate both proliferation and differentiation of kératinocytes

Transforming growth factor -fi and -^2

Transforming growth factor and -P 2 (TGF-Pi and TGFP2) are growth factors that generally suppress the proliferation of epithelial cells (Pietenpol et al. 1990). The putative mechanism is thought to involve the regulation of the retinoblastoma gene product (pRB) function by TGF-P (Pietenpol et al. 1990). This is supported by evidence indicating that events that compromise the function of the pRB protein (ie the presence of simian virus 40 or human papillomavirus oncogenes) uncouple TGF-Pi regulation of proliferation. TGF-P 1 suppresses keratinocyte proliferation but also induces differentiation (Coffrey et al. 1988).

49 Interferon-y Interferon-y (IFN-y) suppresses keratinocyte proliferation. Treatment of keratinocyte cultures with IFN-y promotes an increase in the level of markers of keratinocyte differentiation (ie comifin and transglutaminase type 1) coupled with a concomitant reduction of two growth regulatory gene products, cdc2 and E2F-1 (Saunders and Jetten 1994). TGF-Pi and retinoic acid suppress the IFN-y dependent increase in transglutaminase type 1 and comifin, but do not prevent the growth arrest of cells (Saunders and Jetten 1994).

Vitamin A, Vitamin D and Glucocorticoids Retinoids Retinoids (vitamin A and related ligands) are potent modulators of keratinocyte differentiation. In vitro retinoids regulate a variety of genes in a coordinate manner (Agarwal and Eckert 1990; Agarwal et al. 1991, 1993). Studies in which marker gene expression is used as a measure of differentiation state indicate that retinoids are potent suppressors of keratinocyte differentiation in vitro (Agarwal and Eckert 1990; Agarwal et al. 1991, 1993). In vivo, retinoic acid causes epidermal thickening, stratum granulosum thickening, and parakeratosis and increases the number of cell layers staining for transglutaminase, involucrin, and filaggrin, while loricrin expression is reduced or absent (Rosenthal et al. 1992). Expression of suprabasal keratins K1 and KIO and basal keratin K14 is not changed by retinoid treatment (Rosenthal et al. 1992). Vitamin D

The active form of vitamin D, 1,25-dihyroxyvitamin D 3 [l,25-(OH)2D3], is a potent inhibitor of in vitro keratinocyte proliferation (Chen et al. 1995), although this response is dependent on culture conditions and l,25-(OH)2D3 concentration (Bollag et al. 1995; McLane et al. 1990). 1,25-dihyroxyvitamin D 3 stimulates keratinocyte differentiation in a concentration dependent manner, by enhancing formation of comified envelopes and activation of marker gene expression (Chen et al. 1995). In stratifying keratinocyte cell culture models using de-epidermolysed dermis as the growth substrate, vitamin D stimulates the intermediate steps in keratinocyte differentiation, resulting in increased terminal differentiation and decreased thickness of intermediate cell layers (Regnier and Darmon 1991). This response is due to an increased rate of conversion of suprabasal cells to comeocytes and not to effects on cell proliferation rate (Regnier and Darmon 1991). The biologically active form of vitamin D, 1,25-(0H)2D3 and related ligands are thought to operate via binding to specific

50 vitamin D receptors in the nucleus (Haussier et al. 1988), which are expressed in all skin layers (Milde et al. 1991), although some effects may be mediated via G proteins, or effects on phosphoinositide metabolism or effects on protein kinase C (Studzinski et al. 1993). Hydrocortisone Hydrocortisone enhances keratinocyte differentiation and promotes colony expansion of cultured kératinocytes (Rheinwald and Green 1975). In cell lines derived from squamous cell carcinoma, hydrocortisone has been shown to increase envelope competence and involucrin levels (Cline and Rice 1983), an effect that is antagonised by retinoids (Cline and Rice 1983). Thus the major effect of hydrocortisone and other glucocorticoids is to enhance keratinocyte differentiation.

Calcium A calcium gradient is known to be present in the epidermis with relatively low concentrations of calcium in the basal layer and higher concentrations in the suprabasal layers (Malmqvist et al. 1984; Menon et al. 1985). The presence of this in vivo gradient suggests a role for calcium in regulating keratinocyte differentiation. This possibility is in agreement with in vitro experiments which show that maintaining cultured epidermal kératinocytes in low calcium containing medium (<0.1 mM) results in low level expression of differentiation marker genes, a state which can be reversed by addition of increasing extracellular calcium concentrations (Yuspa et al. 1989). The presence of the gradient in vivo and the calcium-dependent regulation of the genes in vitro provide evidence for a central role for calcium as a regulator of differentiation.

Role of protein kinase C signalling cascade in regulating keratinocyte differentiation Protein kinase C (PKC) consists of a family of 11 isoenzymes, 5 of which are expressed in epidermal kératinocytes (Dlugosz et al. 1992). Physiological activation of PKC occurs when extracellular stimuli activate phospholipase C (PLC), which hydrolyses membrane-localised phosphotidylinositol to produce inositol triphosphate and 1,2-diacylglycerol (DAG). Phosphotidylinositol is involved in mobilising intracellular calcium and DAG is an activator of PKC (Nishizuka 1992). In human kératinocytes, PKC activation increases expression of suprabasal markers of differentiation (Dlugosz and Yuspa 1993). Differentiation can be blocked by inhibitors of PKC function (Bollag et al. 1993; Dlugosz and Yuspa 1993), suggesting that this pathway is required for differentiation.

51 Hair

Hair follicle morphogenesis The hair follicle develops from the embryonic epidermis as an epithelial finger (Fig 1.5). This peg differentiates into three enclosed epithelial cylinders. The central most cylinder forms the hair shaft. The outermost cylinder forms the outer root sheath (ORS) that separates the whole structure from the dermis. The middle cylinder, the inner root sheath (1RS) moulds and guides the shaft in its passage outwards. Hair follicle morphogenesis is different from hair follicle regeneration in mature tissues (Fig 1.5). Follicle formation usually occurs once in the lifetime of an individual, so a mammal is bom with a fixed number of follicles, which does not normally increase thereafter. For recent reviews see Cotsarelis (1998); Millar (2002); Fuchs and Raghavan

(2002).

Hair follicle cycle The hair follicle is a regenerating system (Silver and Chase 1977). All mature follicles undergo a growth cycle consisting of phases of growth (anagen), regression (catagen), rest (telogen) and shedding (exogen) (Stenn and Paus 2001) (Fig 1.5). The anagen follicle consists of a so-called ‘permanent’ portion above the muscle insertion and a cycling portion below. During the regression phase of the hair cycle, the lower part of the hair follicle undergoes programmed cell death (Cotsarelis 1997). Cyclical growth of hair continues throughout postnatal life, and allows the follicle to remodel itself. This is particularly evident in the response of hair follicles to androgens, which cause enlargement of beard hair follicles in adolescent boys, and miniaturization of scalp follicles in men with androgenetic alopecia (Hamilton 1942).

52 / i

R # T elo g en

Figure 1.5; Follicular morphogenesis and hair cycling. The hair cycle is envisioned here as a ‘number 6 \ where the ‘limb’ of the number represents the morphogenetic phases and the ‘circular base’ represents the repeating cycle after morphogenesis. Although the morphogenetic limb occurs just once in the lifetime of the follicle, the cycle repeats many times. DP= dermal papilla, SG = sebaceous gland, APM= arrector pili muscle, HS= hair shaft, mel= melanin, BM=basement membrane, POD= programmed organ deletion, HF=hair follicle, ORS=outer root sheath, IRS=inner root sheath. Taken from Stenn and Paus (2001).

Structure and properties of the hair follicle The mature hair follicle is a complex structure composed of several concentric cylinders of epithelial cells (known as root sheaths), which surround the hair shaft (Sperling 1991), (Fig 1.6).

53 CTX

ORS -CTS

4 ^ — Bulb Matrix

• ' 'tiâ*

Club W ORS m 0-:: Anag

m m m Figure 1.6: Human hair follicle structure. The eight cellular lineages of the mature follicle are shown above. The defined lineages are the outer root sheath (ORS), the companion layer (CL), the internal root sheath Henle's layer (He), internal root sheath Huxley's layer (Hu), the cuticle of the internal root sheath (Csth), the cuticle of the hair shaft (Csft), the cortex of the shaft (CTX), and the medulla of the shaft (Med). The shaft and the 1RS move up the follicle as a unit, with the 1RS falling from the shaft just below the sebaceous gland duct. The slippage plane for the outward-moving shaft sheath is in the companion layer, (a) Vertical section of proximal anagen follicle, low power, (b) Vertical section of proximal anagen follicle, high power, (c) Cross section of proximal anagen follicle, (d) Telogen shaft with anagen follicle below Note the secondary hair germ epithelium at the base of the telogen follicle (arrow). CTS, connective tissue sheath; SG, sebaceous gland; Club, telogen shaft base; Anag, proximal anagen follicle; FP, follicular papilla; GE, germinative epithelium. Taken from Stenn and Paus (2001).

54 The cells of the lower portion of the hair follicle bulb are undifferentiated matrix cells. These are rapidly dividing cells which give rise to eight different cell lineages (Fig 1.6, 1.7). From within outwards, these include the medulla, cortex, and hair cuticle cell lineages which make up the hair shaft; the inner root sheath cuticle, Huxley’s and Henle’s layers which make up the inner root sheath (1RS); the companion layer and the outer root sheath (ORS) (Niemann and Watt 2002).

Hair shaft L

O Ü ^ (a) to o

Hair shatl 1RS 1» Precortex

ORS

J i i :

Matrix Derm al papilta

Figure 1.7: Hair follicle lineages. (a) Haematoxylin- and eosin-stained section through the base of a growing follicle, (b) The different hair follicle lineages; the dermal papilla is shown in dark grey and the hair matrix in light grey. Abbreviations: 1RS, inner root sheath; ORS, outer root sheath. Bar, 50pm. Taken from Niemann and Watt (2002).

Although it is largely epithelial in origin, the follicle contains at its base a ball of specialized dermal cells, the dermal papilla, which play a crucial part in the regulation of successive cycles of postnatal hair growth (Fig 1.7). There are several hypotheses to account for how hair follicles regenerate:

55 Bulge activation hypothesis One view is that there is a single location of hair follicle stem cells, namely the bulge. At the end of each hair growth cycle, the specialized mesenchymal cells of the dermal papilla at the base of each follicle come into close proximity with the bulge, triggering the bulge stem cells to divide and generate progenitors of all the differentiated cells in the growing follicle (Cotsarelis et al. 1990; Fuchs et al. 2001).

Stem cell migration hypothesis Alternatively, stem cells themselves might exit the bulge and migrate along the ORS to the base of the follicle, where they surround the dermal papilla, forming the matrix (Oshima et al. 2001; Fuchs et al. 2001).

Hair follicle predetermination model A different view is that there are two stem cell populations in the hair follicle, one in the bulge and one at the base of the follicle in the hair matrix (Panteleyev et al. 2001). The hair shaft and inner root sheath lineages would then be derived from the matrix stem cell population, whereas the bulge stem cells would give rise to the ORS.

Commitment to the formation of the three hair shaft lineages (medulla, cortex, cuticle) and the three 1RS lineages (cuticle, Huxley’s layer and Henle’s layer) takes place within the hair matrix, and there is a common matrix progenitor cell for the hair shaft lineages and one for the 1RS lineages (Niemann and Watt 2002). The ORS represents a separate lineage and the companion layer may represent the fourth and outermost layer of the 1RS (Niemann and Watt 2002). Stem cell progeny either remain as stem cells or they leave the stem cell compartment, and select a particular differentiation pathway, leading ultimately to irreversible loss of proliferative ability. There are several signalling molecules which regulate the balance between proliferation and differentiation in postnatal hair follicles, of which Sonic hedgehog (Shh) and bone morphogenetic proteins (Bmps) are of central importance. Shh promotes proliferation and Bmps promote differentiation (see Niemann and Watt (2002) for a review).

56 Skin Embryology and Development

The skin of the human embryo begins to develop soon after conception, but the initial simplicity of this single layered epithelium is short lived with the establishment of the epidermis and the dermis within the first 2 weeks of embryonic life. The dermis then lags behind the epidermis in attaining its definitive state. By the end of the second trimester, the epidermis is a keratinised, stratified, squamous epithelium, which includes all of the immigrant cell types and has formed all of its adnexal structures. The dermis, at the same stage is less than half its full thickness and still immature (Holbrook 1991). The epidermis The epidermis is an epithelium of ectodermally derived cells (kératinocytes), which, during the course of development, receives additional cells (immigrant cells) from other germ layers. Localised proliferations and down-growths of basal kératinocytes form the appendages: pilosebaceous structures, eccrine sweat glands, apocrine glands and nails. Epidermal stratiHcation The primitive epidermis is established at 7-8 days when ectoderm and endoderm are defined in the inner cell mass of the implanted blastocyst. At this stage the epidermis is a single-layered ‘indifferent ectoderm’. A second epidermal layer forms at the end of the fourth week. The outermost of the two layers is the periderm. The periderm is the transient, protective covering of the epidermis that is sloughed into the amniotic fluid as soon as differentiation of the underlying epidermal layers is complete at 20-24 weeks. Beneath the periderm, basal kératinocytes comprise the single layer of the ‘epidermis proper’. At the end of the ninth week, the embryonic-fetal transition, basal cells divide to give rise to daughter cells that move vertically to form the first intermediate cell layer that characterises the fetal epidermis. Two or three more layers of intermediate cells are added to the epidermis in the second trimester (12-24 weeks) of life. These cells lie between periderm and basal layers. Once a granular layer is established in the sixth month, the intermediate cells become known as spinous cells; thus each layer of the epidermis assumes the adult nomenclature as the tissue assumes the adult characteristics. The first stratum comeum develops at the end of the second trimester (24 weeks). At first it consists of only a few cell layers, but it increases in thickness during the third trimester and, at birth is approximately equivalent to that of the adult.

57 The periderm The periderm is the transient, protective covering of the epidermis that is sloughed into the amniotic fluid as soon as differentiation of the underlying epidermal layers is complete. The periderm undergoes a series of morphologic changes that may affect the function of the skin prior to kératinisation (Holbrook 1991). The periderm of the embryo is a pavement epithelium of small polygonal cells that are modified at the amniotic surface by microvilli and a glycocalyx (Hoyes 1967). The cells join at their apical borders by tight junctions that completely encircle the cell and may block intercellular seepage of contents from the amniotic sac into the developing skin. Periderm cells increase in volume and double in diameter at the time the first intermediate layer is formed. Towards the end of the first trimester and into the fourth month, periderm cells develop a central bleb, still covered with microvilli and a cell coat that extends into the amniotic fluid. The blebs are responsible for the designation of periderm cells as ‘bladder cells’. They are filled with glycogen, may contain the nucleus and are reinforced peripherally with actin microfilaments. The blebs appear to detach into the amniotic fluid (Nieland et al. 1970; Bergstrom 1979). The surface of each periderm cell may simultaneously exhibit microplicae and foliate and rod-like microvilli. These surface modifications increase the area of the plasma membrane exposed to the amniotic fluid and suggest either that the periderm cells allow cellular transport from the amniotic fluid into the cell or that the fetal epidermis may also contribute to the amniotic fluid. In the fifth and sixth months of fetal development kératinisation proper of the epidermis occurs and the periderm becomes restricted to a thin layer; microvilli decrease in number and the blebs collapse and persist as shrunken.remnants. The contents of the periderm cell change correspondingly; organelles undergo partial or complete degeneration, glycogen is gone, filaments predominate in the cytoplasm, and the plasma membrane retains a cell coat but also acquires a band of material identical to the involucrin-rich comified cell envelope of the terminally differentiated keratinocyte. Many of the changes that occur in the periderm cell are morphologically indicative of cell death, however they are similar to the changes that occur to the first intermediate cells that undergo partial kératinisation. It is likely that the initial comification of the periderm cell membrane followed by kératinisation of the epidermis protects the fetus against its immediate environment.

58 The layer of periderm cells becomes disrupted at sites where underlying cells have keratinised. This occurs first over the nail bed, then above the hair canal, at orifices of ducts of the adnexal glands and lastly in the interfollicular regions of the skin (Holbrook and Odland 1978). Desquamated periderm cells accumulate with other cellular debris and sebaceous lipids in the vemix caseosa and float freely in the amniotic fluid in a nearly unchanged state until birth (Holbrook 1991).

59 Cutaneous wound healing

Cutaneous wound healing involves three overlapping phases: inflammation, tissue formation and tissue remodelling (Bello and Phillips 2000). This process of regeneration and repair eventually leads to the formation of a scar. This involves a complex, dynamic series of events including clotting, inflammation, granulation tissue formation, epithelialisation, neovascularisation, collagen synthesis and wound contraction. These activities occur in a carefully regulated and reproducible cascade which is initially triggered by tissue injury (for a review see Glat and Longaker (1997); Singer and Clark (1999)). This thesis will concentrate on wound epithelialisation, so an overview of events in epidermal wound healing is presented here. Wound epithelialisation Re-epithelialisation is the term used to describe the resurfacing of a skin wound with a new epithelium (O'Toole 2001). It involves multiple processes including the formation of a provisional wound bed matrix, the migration of epidermal kératinocytes from the cut edge of the wound, the proliferation of kératinocytes that feed the advancing and migrating epithelial tongue, the stratification and differentiation of new epithelium and the reformation of the basement membrane zone. Immediately after a wound occurs, there is a short lag period lasting for several hours. The kératinocytes that form the cut edge of the wound begin to migrate at 24-48 hours. The kératinocytes migrate in not only from the wound periphery, but also from cut epidermal appendages throughout the wound bed. Migrating kératinocytes change phenotype Electron microscopy studies of epidermal cells at the initiation of migration after wound healing have shown that they change in shape as they go from being a stationary basal keratinocyte to a migrating cell. They become flat and elongated and develop long cytoplasmic extensions called lamellipodia (Odland and Ross 1968). The migrating cell loses its intercellular desmosomes (which provide physical connections between the cells), and gap junctions become more prominent (Goliger and Paul 1995). Keratin filaments retract from the cytoplasmic periphery and there is redistribution of the actin cytoskeleton into lamellipodia, which allow cell movement (O'Toole 2001). Model of keratinocyte motility The mechanisms of keratinocyte motility are not known, but a current model is that a keratinocyte is thought to migrate via a series of ‘ratchet’ like movements. The

60 adherent cell extends its lamellipodia, forms attachments to the underlying wound matrix, assembles and contracts its cytoskeleton and finally, as it moves forwards, disengages matrix adhesions by expression of matrix metalloproteinases and other proteinases that degrade the wound matrix (O'Toole et al. 1997). Kératinocytes dissect the wound Hemidesmosomal links between the epidermis and the basement membrane are lost, and this allows lateral movement of kératinocytes. The expression of integrin receptors on kératinocytes allows them to interact with a variety of extracellular matrix proteins (e.g. fibronectin and vitronectin) that are interspersed with stromal type I collagen at the margin of the wound and interwoven with the fibrin clot in the wound space (Clark 1990; Larjava et al. 1993; Clark et al. 1996). The migrating kératinocytes dissect the wound, separating desiccated eschar from viable tissue. The path of dissection appears to be determined by the array of integrins that the migrating kératinocytes express on their cell membranes. Kératinocytes produce collagenase and plasminogen activator (which activates plasmin) in order to degrade the extracellular matrix to allow cell migration between the collagenous dermis and the fibrin eschar (Bugge et al. 1996; Pilcher et al. 1997). Kératinocytes at the wound margin proliferate One to two days after injury, kératinocytes at the wound margin begin to proliferate behind the actively migrating cells. The stimuli for migration and proliferation of kératinocytes during re-epithelialisation have not been determined, but several possibilities exist. The absence of neighbouring cells at the edge of the wound margin (the ‘free edge’ effect) may signal both migration and proliferation. Local release of growth factors and increased expression of growth factor receptors may also stimulate these processes (e.g. via epidermal growth factor (EOF), transforming growth factor a (TGFa), and keratinocyte growth factor (KGF)) (Singer and Clark 1999). As re- epithelialisation ensues, basement membrane proteins reappear in a very ordered sequence from the margin of the wound inwards (Clark et al. 1982). Kératinocytes revert to their normal phenotype, once again firmly attached to the re-established basement membrane and underlying dermis.

61 Skin cancer

Skin cancers are the commonest malignancies and the incidence is steadily increasing. Skin cancers account for 1% of all cancer deaths (American Cancer Society 1992). The prevalence of skin cancer and the external location of the lesions provide an excellent opportunity for studying the factors regulating cancer induction in skin. As a result, more is known of the aetiology, the influence of host susceptibility factors and the characteristics of preneoplastic and neoplastic lesions than of any other human malignancy. There are three main types of skin cancer: basal cell, squamous cell and melanoma. Non-melanoma skin cancer Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (SCC) are the commonest malignancies in white people, accounting for greater than 95% of non melanoma skin cancers (NMSC) (Miller and Weinstock 1994). An estimated 2.75 million cases are diagnosed per year world-wide (Strom and Yamamura 1997), with approximately one million cases per year diagnosed in the USA (Miller and Weinstock 1994). It is a very common problem and seems to be increasing in incidence as lifestyles and fashions for sun-bathing have changed during the twentieth century. BCCs and SCCs are both cutaneous malignancies arising from the same cell type, the keratinocyte, but they are different tumours with different properties.

Aetiology Ultraviolet radiation Exposure to ultraviolet radiation (UV) correlates directly with the development of skin cancer (Stal and Spira 1997). Epidemiological studies have established the importance of UV radiation exposure in BCC and SCC tumorigenesis. NMSCs are found predominantly on areas of skin exposed to the sun, primarily the head and neck

(approximately 6 6 %) (Giles et al. 1988). They occur with greater frequency in those with fair skin, an inability to tan, and a tendency to freckle or sunburn (Kricker et al. 1991). UVB radiation appears to be the most important aetiological factor; UVA plays a minor role. UV radiation is known to damage the DNA of kératinocytes (Livneh et al. 1993). Radiation induced damage to DNA is responsible for cell death and neoplastic transformation.

62 Immune suppression People with immune suppression experience higher rates of cancer than the general population. Excessive ultraviolet radiation interferes with the normal functioning of the immune system and compromises the immunologic defences of the skin (for a review see (Beissert and Schwarz 1999)). Other causes of immunosuppression include: persons with inborn defects in immunity, organ transplant recipients and HIV-infected persons (Mueller 1999). Most of the excess malignancies seen in these populations are those that are associated with oncogenic viral infections. Non-melanoma skin cancer is especially common in transplant recipients taking long term immunosuppressive drugs. This group have a cumulative incidence of non melanoma skin cancer of up to 40% after 15 years of immunosuppression (Glover et al. 1993). Non-melanoma skin cancers in organ transplant recipients may be caused by loss of immune control of oncogenic human papilloma virus (HPV) infections. The evidence for this is discussed in the section on HPV.

Basal cell carcinoma Basal cell carcinoma is the most common skin cancer, accounting for approximately 70% of all cutaneous malignancies and the incidence is rising. They are five times more common than squamous cell carcinomas. The main differences between BCCs and SCCs are that in BCCs, metastasis is very rare and also BCCs have no clear precursor lesions, unlike SCCs (Miller 1991a, 1991b). It is believed that the origin of the BCC is the outer root sheath of the hair follicle (Asada et al. 1993). Genetic abnormalities and mutations are thought to play a major role in the development of BCC, especially in inherited syndromes of BCC. Patients with xeroderma pigmentosum cannot repair the UV-induced DNA mutations and have an increased risk for cutaneous carcinogenesis. Studies on patients with nevoid basal cell carcinoma syndrome (Gorlin’s syndrome) have shown mutations of the PTC (PATCHED) gene in BCCs arising in these patients (Hahn et al. 1996; Johnson et al. 1996). This gene is a tumour suppressor gene that plays a key role in the regulation of the Sonic Hedgehog signalling pathway that stimulates growth (Johnson et al. 1996). UV-induced p53 tumour suppressor gene mutations are also seen in more than 50% of BCCs (Ziegler et al. 1994). There is considerable variability in the morphology of basal cell carcinomas, and several histopathological subtypes have been defined (for a review see Rippey (1998)). However, BCCs share certain features: they are composed of islands or nests of basaloid

63 cells, with palisading of the cells at the periphery and a haphazard arrangement of cells at the centre. Tumour cells have a hyperchromatic nucleus with relatively little, poorly defined cytoplasm. There are numerous mitotic figures, sometimes atypical, and a correspondingly high number of apoptotic tumour cells. BCCs can be locally invasive and reach a large size.

Squamous cell carcinoma Cutaneous squamous cell carcinomas represent malignant epithelial neoplasms with the ability to infiltrate locally and destroy tissue. Initially developing as carcinoma in situ, tumours later become invasive, involving dermis, subcutis, musculature, cartilage or bone and may lead to regional lymph node disease and distant metastatic spread (Fetter and Haustein 2000). They are the second commonest skin cancers in the white population and the commonest form of skin cancer in black people (Gloster and Brodland 1996). Most squamous cell carcinomas (SCCs) arise in areas of direct exposure to the sun, with non-exposed areas occasionally affected. Uncommonly SCC may develop at sites of chronic ulceration, trauma, bums, frostbite, vaccination scars, fistula tracts, pilonidal sinuses of long standing. Other aetiological agents include radiation therapy, arsenic, coal tar and various hydrocarbons. Individuals with genodermatoses such as xeroderma pigmentosum, albinism and epidermodysplasia verruciformis are at increased risk for SCCs (Fetter and Haustein 2000). Mutations of the p53 tumour suppressor gene also occur in SCCs (Brash et al. 1991). The development of SCC does not occur by a simple single step, but rather is thought to be a multistage process (Grossman and Leffell 1997). Conversion of susceptible kératinocytes to premalignant cells and then progression to carcinoma occurs as a result of successive genetic hits (Diem and Runger 1997). In the initiation stage of carcinogenesis, there is clonal expansion of premalignant cells. The next stage is increased proliferation of premalignant cells and subsequent chromosomal aberrations. The final stage is conversion to SCC (Bagheri and Safai 2001). There are clinicopathological variants of cutaneous SCCs (for a review see Lohmann and Solomon (2001)). Histologically, SCCs usually consist of nests of squamous epithelial cells, which arise from the epidermis and extend into the dermis for a variable distance. The cells have abundant eosinophilic cytoplasm and a large, often vesicular, nucleus. There is variable central kératinisation and horn pearl formation, depending on the differentiation of the tumour. SCCs sometimes infiltrate along nerve sheaths, the adventitia of blood vessels, lymphatics, fascial planes and embryological

64 fusion planes. They may evoke a strong desmoplastic response. There is often a mild to moderate chronic inflammatory cell infiltrate at the periphery of the tumour. The differential diagnosis of a SCC includes: keratoacanthoma, pseudoepitheliomatous hyperplasia, inverted follicular keratosis, basal cell carcinoma with squamous metaplasia and adnexal carcinoma. These must all be ruled out. It has been shown that the risk of metastatic spread significantly increases as differentiation deteriorates and the vertical tumour thickness increases (Fetter and Haustein 2000).

65 Human papilloma virus

Human papillomaviruses (HPVs) are small double-stranded DNA viruses that are widespread in the human population. They are strictly epitheliotropic and infect only cutaneous and mucosal skin sites. Over 120 different HPV types have been identified, of which 80 have been characterised in full (de Villiers et al. 1997) and the most relevant types are shown in the table below

Table 1.4: HPV types and their associated disease states. HPV type Site Associated disease

1,2 Cutaneous Verruca plantaris

5, 8 Cutaneous Epidermodysplasia verruciformis (Squamous cell carcinoma)

6 , 11 Genital mucosa CINI and n Condyloma acuminata Laryngeal papilloma 3,10,15,41 Cutaneous Epidermodysplasia verruciformis (benign) 16, 18, 31, 33, 45, 51 Genital mucosa CIN I, U, and m Cervical carcinoma

HPV infections are classified into cutaneous, cutaneous involved in epidermodysplasia verruciformis (EV), cutaneous and mucosal, and mucosal of low and high risk (Majewski and Jablonska 1997). HP Vs are linked with cervical cancer and squamous cell carcinoma of the anus (for a review see zur Hausen (2002)), which are associated with high risk genital HPV types 16, 18, 31, 33 and the low risk genital HPV types 6 and 11 (Gissman et al. 1984). The best-studied association of HPV with malignant transformation at cutaneous sites is in the rare skin disorder epidermodysplasia verruciformis (EV). EV patients show an increased incidence of skin carcinoma and HPV-5 and HPV -8 are usually found in those sun-exposed lesions which progress to carcinomas (Orth et al. 1979). These HPV subtypes however are not commonly found in the general population (Tieben et al. 1994).

66 HPV and human non-melanoma skin cancer Human papilloma viruses are increasingly recognised as an important human carcinogen and have been implicated in non-melanoma skin cancers (Proby et al. 1996; zur Hausen 1996). The association between skin warts and skin cancer was first noted in renal transplant recipients (Walder et al. 1971). Renal transplant recipients have a marked increase in susceptibility to both viral warts and non-melanoma skin cancer. The ratio of basal cell carcinomas (BCCs) to squamous cell carcinomas (SCCs) is 5:1 in immunocompetent populations, whereas in immunosuppressed patients the ratio is reversed, with the risk of developing a SCC up to 250 times greater and the risk of developing a BCC ten times greater than that in the general population (de Villiers et al. 1997). Viral warts and cutaneous SCCs both appear on sun-exposed sites and ultraviolet light seems to be an important factor in the development of both warts and cancers (Glover et al. 1993). In addition, clinical and histological features of transplant squamous cell carcinomas indirectly support the progression of viral warts through increasingly dysplastic squamous lesions to invasive squamous cell carcinomas (Blessing et al. 1989). Until recently it has been technically difficult to detect human papilloma virus in skin cancers. In early studies, detection of HPV DNA varied in both overall prevalence from 0-64% and in the HPV types detected (for a review see Proby et al. (1996)). As a consequence, the true prevalence of HPV in cutaneous lesions was underestimated. A recent study using a degenerate PCR technique has analysed the HPV status of non melanoma skin cancers in both immunosuppressed and immunocompetent individuals (Harwood et al. 2000). They found that a high prevalence of HPV DNA was detected in lesions from immunosuppressed patients (82%), with no significant difference in either HPV prevalence or types between SCCs, BCCs and premalignant lesions. In immunocompetent patients, the overall HPV DNA prevalence of 36% was significantly lower (P<0.001). Epidermodysplasia verruciformis HPV types predominated in all tumours but in renal transplant patients, co-detection of multiple HPV types within single lesions occurred. The precise relationship between HPV and the initiation of carcinogenesis remains unclear. Several possible models exist (Harwood et al. 2000). Viruses may have a promoter effect, acting in conjunction with specific tumour initiators, the most important being ultraviolet radiation. Alternatively, HPV may simply be a cutaneous “passenger”. HPV DNA is also detected in normal skin. In one study, 16% of normal skin samples from individuals with non-melanoma skin cancer were found to contain

67 HPV (Stark et al. 1994). The majority of hair follicles from immunosuppressed patients were also found to harbour HPV DNA (Boxman et al. 1997). A high prevalence of HPV DNA has also been found in the benign proliferative skin condition psoriasis (Favre et al. 1998; Weissenbom et al. 1999). It is possible that immunosuppression and ultraviolet light may both increase susceptibility to cutaneous viral infection (or activation of latent HPV infection) and skin cancer independently. Alternatively, a hyperproliferative or premalignant epidermis in the permissive milieu of systemic immunosuppression may preferentially enhance the replication of HPV.

68 III. ROLE OF PURINES IN SKIN

Purinergic signalling plays important roles in both short-term, fast actions and long-term, slower, trophic actions (Bumstock 1997). Examples of each process that are relevant to the skin are discussed below.

Short term purinergic signalling

Purinergic signalling in cutaneous blood vessels Purines have a role to play in control of cutaneous vasculature tone. ATP, ADP and AMP have vasodilatory effects on the artery supplying canine facial and nasal vascular beds when infused intra-arterially (Bari et al. 1993). ATP is released with noradrenaline from perivascular sympathetic nerves (Kugelgen and Starke 1985; Kennedy et al. 1986). ATP is also released from endothelial cells during changes in

flow (shear stress) and hypoxia to act on P2Yi and P 2 Y2 receptors, leading to the production of nitric oxide and subsequent vasodilatation (Bumstock 2002a). Vasodilator P2Y and PI purinoceptors and vasoconstrictor P2X purinoceptors in human subcutaneous resistance arteries have been identified (Martin et al. 1991). They could have an important role to play in wound healing.

Long term purinergic signalling

Purinergic signalling in the epidermis Contradictory effects of purine nucleotides on keratinocyte proliferation have been previously reported. Adenosine triphosphate (ATP) at concentrations of l-lOOpM have been demonstrated to inhibit terminal differentiation and to stimulate thymidine incorporation in keratinocyte cultures (Pillai and Bikle 1992). Other investigators not using optimal cell culture conditions, for example elevated calcium levels that promote keratinocyte differentiation and quiescence as well as significant fibroblast contamination, have suggested that adenosine, deoxyadenosine and adenine nucleotides can inhibit the epidermal outgrowth from both human and porcine kératinocytes (Harper et al. 1974; Flaxman and Harper 1975; Taylor et al. 1980; lizuka et al. 1984). A study tried to unravel this and established that proliferation of keratinocyte cultures was inhibited by exogenous adenosine, AMP, ADP, ATP and ATPyS at submillimolar concentrations (Cook et al. 1995). This inhibition occurred in the presence or absence of EOF and adenosine had the most potent inhibitory effect.

69 PI receptors

Human kératinocytes express A 2B receptor mRNA but not significant levels of

Ai, A2A or A 3 receptor mRNA (Brown et al. 2000). This group showed that adenosine,

AMP and A2 adenosine receptor agonist, NECA, induced elevation of cAMP in kératinocytes (Brown et al. 2000). However, adenosine and adenine nucleotides inhibited normal and transformed keratinocyte proliferation but NECA was not a potent inhibitor of keratinocyte proliferation This led them to conclude that the anti proliferative action of adenosine may not be mediated through A 2B purinoceptor activation and subsequent elevation of cAMP. To determine whether this was the case, they then tested to see if the A1/A2 class purinoceptor antagonist, theophylline, could reverse the antiproliferative effect of adenosine. Theophylline attenuated the ability of adenosine and AMP to elevate intracellular cAMP, but theophylline had no detectable effect on the concentration-dependent adenosine mediated growth inhibition of keratinocyte cultures. However, dipyridamole (a nucleoside transport blocking agent) inhibited the transport of adenosine into kératinocytes and reversed the anti-proliferative effect of adenosine and adenine nucleotides. They then suggested that the mechanism for adenosine mediated growth inhibition was not via an A 2B receptor but via a mechanism involving adenosine transport into the cell.

P2 receptors The presence of P2 purinergic receptors was suggested by a study on canine kératinocytes (Suter et al. 1991). Extracellular ATP stimulates P2 purinergic receptors to transiently increase intracellular calcium levels. An increase in intracellular calcium in cultured human kératinocytes is an early event in terminal differentiation (Sharpe et al. 1989), which is of primary importance during that process. In the presence of l.SmM extracellular calcium, 100 and SOOpM ATP caused a rapid three to twelve fold rise in intracellular calcium levels in canine kératinocytes above resting levels. UTP was as effective as ATP in stimulating rises in intracellular calcium levels. P2X receptors

P2 X5 and P2Xv receptors have been localised using immunohistochemistry in rat epidermis. P2Xg receptors were expressed on cutaneous kératinocytes undergoing proliferation and differentiation, while P2X? receptors were associated with keratinised dead cells. Functional roles in the regulation of proliferation, differentiation and cell death were proposed (Groschel-Stewart et al. 1999a).

70 P2Y receptors

ATP induced production of IP 3 and elevation of intracellular calcium have been reported in keratinocyte cultures (Pillai and Bickle 1992), suggesting that these cells

may express P2Y receptors. P 2 Y2 receptor mRNA has been localised in human epidermal basal cells via in situ hybridisation (Dixon et al. 1999), in human retinal pigment epithelial cells (Sullivan et al. 1997), equine epithelial cells (Wilson et al.

1998), and frog skin epithelium (Brodin and Nielsen 2000). The potent P 2 Y2 receptor agonist, UTP has also been shown to cause proliferation in kératinocytes (Dixon et al. 1999) and HaCaT cells (Lee et al. 2001).

Purinergic signalling in cutaneous wound healing

Topically applied adenosine Ai or A 2A receptor agonists promotes cutaneous wound healing in full thickness excisional wounds in normal and diabetic mice and rats (Montesinos et al. 1997; Sun et al. 1999). Evidence from experiments performed on adenosine A 2A receptor knockout mice demonstrates that the A 2A receptor is the adenosine receptor subtype involved in wound healing (Montesinos et al. 2002). This group found that exogenous adenosine A 2A receptor agonists increased angiogenesis in healing wounds and endogenously released adenosine interacted with adenosine A 2A receptors to promote angiogenesis in an internal wound (murine air pouch model) (Montesinos et al. 2002). Signalling via ATP has also been implicated in wound healing (Abbracchio and Bumstock 1998), but the effect of ATP in cutaneous wound healing has not yet been examined. Both ATP and UTP promoted healing of wounds made by mechanically denuding areas in confluent monolayers of renal epithelial cells (Sponsel et al. 1995). Stimulation of wound healing by ATP was equivalent in control cells and in cells in which irradiation suppressed proliferation, suggesting a prominent role for cell migration in the healing process. Specific experiments with phorbol esters and protein kinase C inhibitors suggested a role for this enzyme in adenine nucleotide stimulation of wound healing. Propagation of cell injury signals has also been shown to depend highly on activation of extracellular P2 receptors. In rat liver epithelial cells (WB-F344 and WB- aBl cells), mechanically induced cell injury initiated signalling through at least two pathways, one involving intercellular communication via gap junctions, and the other involving extracellular communication via activation of P2 receptor linked to Ca^"^ wave propagation (Frame and de Feijter 1997). Treatment of cells with the P2 receptor

71 blocker suramin significantly reduced both the rate and extent of Ca^^ wave propagation in WB-F344 and completely blocked its propagation in WB-aBl cells. These data would suggest a role for nucleotides in both the early intercellular communication of damage as well as in the consequent long-term changes leading to healing and recovery.

72 Project aims

The overall aim of this thesis was to establish whether purinergic receptors have a functional role in the homeostasis, tissue regeneration, fetal development and pathophysiology of the epidermis of human skin and whether these roles may have therapeutic implications. Specific aims were to examine:

1. The distribution of purinergic receptor subtypes within normal adult epidermis using immunohistochemistry. 2. Whether purinergic receptors have a functional role in human keratinocyte proliferation by means of an in vitro proliferation assay using different known agonists and antagonists of purinergic receptor subtypes. 3. The distribution of purinergic receptor subtypes within anagen hair follicles. 4. The distribution of purinergic receptor subtypes within human fetal epidermis. 5. Whether the distribution of purinergic receptors changes during wound healing using different model systems of normal and delayed wound healing in rat skin and to quantify those changes in receptor expression using optical densitometry.

6 . The distribution of purinergic receptors within different types of non-melanoma skin cancers. To establish whether purinergic receptors in non-melanoma skin cancer are functional by using an in vitro culture model with a cutaneous squamous cell carcinoma cell line, A431, and to define their pharmacology in terms of purinergic receptor subtype agonists and antagonists. 7. The expression of purinergic receptors in cutaneous warts and in a raft organotypic human papillomavirus culture model.

73 RESULTS

74 SECTION A NORMAL TISSUES

75 CHAPTER 2 PURINERGIC RECEPTORS ARE PART OF A FUNCTIONAL SIGNALLING SYSTEM FOR PROLIFERATION AND DIFFERENTIATION OF NORMAL HUMAN EPIDERMAL KERATINOCYTES

76 ABSTRACT

The expression of P2Xs, P2X?, P2Yi and P 2 Y2 receptor subtypes was investigated in normal human epidermis and in relation to markers of proliferation (PCNA and Ki-67), keratinocyte differentiation (cytokeratin KIO and involucrin) and markers of apoptosis (indicative of keratinocyte terminal differentiation - TUNEL and anti-caspase-3). Using immunohistochemistry, it was shown that each of the four receptors was expressed in a spatially distinct zone of the epidermis, suggesting different functional roles. Functional studies were performed on primary cultures of human kératinocytes and on explanted rat skin, where different purinergic receptor subtype agonists and antagonists were applied to cultured kératinocytes or injected subcutaneously into the skin, respectively. An increase in cell number was caused by low doses of the non-specific P2 receptor agonist ATP, the P 2 Y2 receptor agonist UTP (P<0.001), and the P2Y% receptor agonist 2MeSADP (P<0.05). There was a significant decrease in cell number as a result of treatment with high doses of adenosine (P<0.001) and ATP (P<0.001), the P2Xs receptor agonist ATPyS (P<0.001) and the P2X? receptor agonist BzATP (P<0.001). Suramin caused a significant block in the effect of lOOpM ATP (P<0.01) and lOOOpM ATP (P<0.001) on cell number. The effect of high doses of adenosine and ATP was not changed by the A 1/A2 adenosine receptor antagonist, 8 -(p- sulfophenyl)theophylline or the adenosine transport blocking agent, dipyridamole.

These results imply that the effect of adenosine is not mediated via A% or A 2 receptors and that different P2 purinergic receptors have different functional roles in the human epidermis, with P2Y% and P 2Y2 receptors controlling proliferation while P2Xs and P2Xv receptors control early differentiation and terminal differentiation of kératinocytes respectively.

77 INTRODUCTION

Recently, there has been much interest in the biological effects of extracellular purine nucleotides and nucleosides in a variety of cell and tissue types. Adenosine triphosphate (ATP) is now recognised as an important messenger molecule for cell-cell communication, with ATP binding specifically to P2 purinergic receptors (Bumstock 1997; Ralevic and Bumstock 1998). The majority of studies have been concerned with the short-term events that occur in neurotransmission and secretion (Bumstock 1997). Now, there is increasing evidence that purinergic signalling can have long-term, trophic effects in embryonic development, growth and cell proliferation (Abbracchio and Bumstock 1998; Bumstock 2001b, 2002a). Purinergic receptors are classified into two groups: PI receptors are selective for adenosine and P2 receptors are selective for adenosine 5’-triphosphate (ATP) and adenosine 5’-diphosphate (ADP), which act as an extracellular signalling molecules (Bumstock 1978). P2 receptors are divided into two main families: P2X and P2Y, based on molecular stmcture, transduction mechanisms and pharmacological properties (Abbracchio and Bumstock 1994). P2Y receptors are G-protein coupled and the principal signal transduction pathway involves phospholipase C, which leads to the formation of inositol 1,4,5-triphosphate (IP 3) and mobilization of intracellular calcium.

IP3 regulates cell growth and DNA replication (Berridge 1987). Seven subtypes of P2Y receptors have been described so far (King et al. 2000; Communi et al. 2001). In contrast, P2X receptors are ligand-gated ion channels, and are activated by extracellular ATP to elicit a flow of cations (Na"^, K^, and Ca^"^) across the plasma membrane. Seven subtypes of P2X receptors are recognised (Khakh et al. 2001); all P2Xi.? subunits have been cloned from mammalian species and are capable of assembling into homo- or heteromultimeric receptors (Torres et al. 1999). There is growing evidence that ATP may act as an important local messenger in the skin, particularly the epidermis. Previous work has shown that adenosine and ATP have an anti-proliferative effect on cultured human kératinocytes (Cook et al. 1995) and that adenosine A 2B receptor mRNA is present in human kératinocytes (Brown et al. 2000). Brown et al. suggested that the mechanism for adenosine mediated growth inhibition was not via an A 2B receptor but via a mechanism involving adenosine transport into the cell (Brown et al. 2000). P2 purinergic receptors are expressed in rat epidermis, on cutaneous kératinocytes, and functional roles in the regulation of proliferation, differentiation and cell death have been proposed (Groschel-Stewart et al.

78 1999a). In particular, P2Xs receptors are expressed on cells undergoing proliferation and differentiation, while P2X? receptors are associated with keratinised dead cells.

P2 Y2 receptors, found in the basal layer of normal epidermis, are claimed to be involved in keratinocyte proliferation (Dixon et al. 1999). P2Yi receptors are thought to be mitogenic in vascular smooth muscle cells and endothelial cells (Bumstock 2002a). The epidermis is a multilayered squamous epithelium in which dividing basal cells withdraw from the cell cycle and progressively differentiate as they are displaced towards the skin surface. Eventually the cells lose their nuclei and other organelles to become flattened squames, which are finally shed from the surface as bags of cross- linked keratin filaments enclosed in a comified envelope (Leigh et al. 1994). Kératinocytes can undergo apoptosis when stimulated by a variety of agents (McCall and Cohen 1991). There is evidence that the normal differentiation programme uses components of the apoptotic biochemical machinery to produce a comified cell and that these mechanisms are required for the normal loss of the nucleus. Indeed, differentiating kératinocytes and apoptotic cells share features, such as DNA fragmentation (Polakowska et al. 1994) and caspase-3 expression (Weil et al. 1999). This study demonstrates the distribution of P2X and P2Y receptors in human epidermis for the first time. Functional studies were performed on primary cultures of human kératinocytes and on explanted rat skin, where purinergic receptor subtype agonists and antagonists were applied to cultured kératinocytes or injected subcutaneously into the skin, respectively. We show that purinergic receptors on kératinocytes are functional and propose that these receptors are part of the normal homeostatic mechanisms controlling keratinocyte proliferation, differentiation and death.

79 MATERIALS AND METHODS

Tissues Twenty-six samples of normal human skin were examined immunohistochemically in this study. Ethical Committee Approval was obtained to harvest human skin. Samples of post-operatively redundant skin from reduction mammoplasty, abdominoplasty, and from the leg were obtained. Neonatal skin samples came from pre-auricular skin tags and accessory digits. Tissue was frozen in isopentane pre-cooled in liquid nitrogen. Blocks were sectioned at 10pm on a cryostat (Reichert Jung CM1800), collected on gelatin-coated slides and air-dried at room temperature. The slides were stored at -20°C. Preparation of antibodies The immunogens used for production of polyclonal P2X antibodies were synthetic peptides corresponding to 15 receptor-type-specific amino acids (AA) in the intracellular C-termini of the cloned rat P2X receptors. The peptides were covalently linked to keyhole limpet haemocyanin. The peptide sequences are as follows: P 2X5: A A 437-451, RENAIVNVKQSQILH; P2X?: AA 555-569, TWRFVSQDMADFAIL. The polyclonal antibodies were raised by multiple monthly injections of New Zealand rabbits with the peptides (performed by Research Genetics, Huntsville, Ala., USA). The specificity of the antisera had been previously verified by immunoblotting with membrane preparations from cloned P 2X 1.7 receptors expressing CHO K1 cells. The antibodies recognized only one protein of the expected size in the heterologous expression systems, and were shown to be receptor-subtype specific (Oglesby et al.

1999). P 2X5 and P2X? antibodies were provided by Roche Bioscience (Palo Alto, California, USA) and were kept frozen at a stock concentration of 1 mg/ml.

Polyclonal anti-P2Yi and P 2 Y2 antibodies were obtained from- Alomone Labs (Jerusalem, Israel), and corresponded to the third extracellular loop of the P2Yi (AA

242-258) and P 2 Y2 receptor (AA 227-244). Antibodies were kept frozen at a stock concentration of 0.6mg/ml (P2Y%, P 2Y2). Immunohistochemistry For immunostaining of cryostat sections, the avidin-biotin technique was used according to a revised protocol (Llewellyn-Smith et al. 1992, 1993). Air-dried sections were fixed for 2 minutes in 4% formaldehyde in O.IM phosphate buffer, containing 0.2% of a saturated solution of picric acid (pH 7.4). Endogenous peroxidase was

80 blocked for 10 minutes with 50% methanol containing 0.4% hydrogen peroxide. Non

specific binding sites were blocked by a 20 minute pre-incubation in 10% normal horse serum (NHS) in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, Poole, UK), followed by incubation with the primary antibodies diluted to 1:100 or 1:200 in antibody diluent (10% NHS in PBS + 2.5% sodium chloride (NaCl) at 4°C overnight. Subsequently, the sections were incubated with biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA, USA) diluted to 1:500 in 1% NHS in PBS for 30 minutes, followed by ExtrAvidin peroxidase conjugate (Sigma) diluted to 1:1000 in PBS for 30 minutes at room temperature. After a wash step, a nickel- diaminobenzidine (DAB) enhancement technique was used to visualise the reaction product. Sections were washed three times with PBS after each of the above steps except for after pre-incubation with 10% NHS. After the last wash, sections were dehydrated twice in isopropanol and mounted with EUKITT (BDH Laboratory Supplies, Poole, UK). For immunofluorescent staining, sections were fixed and pre-^incubated with 10% NHS in PBS-merthiolate, followed by incubation with the primary antibodies overnight, as above. Biotinylated donkey anti-rabbit IgG antibody (Jackson ImmunoResearch Lab), diluted 1:500 in 1% NHS in PBS, was applied for 1 hour followed by Streptavidin-FTTC (Amersham Life Science), or Streptavidin Texas red (Amersham International pic., UK), diluted 1:200 in PBS-merthiolate for 1 hour at room temperature. Sections were washed in PBS and then mounted in Citifluor (Citifluor Ltd, London, UK). Control experiments were carried out with primary, secondary and tertiary antibodies omitted from the staining procedure or the primary antibodies preabsorbed with the corresponding peptides. For the preabsorption control, 1.5pl of the anti-P2X antibody (at 1 mg/ml) was incubated with 24pl of the respective peptide at 5mg/ml overnight at 4°C. 275pi of NHS was added to give 300pl of a 5pg/ml concentration of

the P2X antibodies. For P2Yi and P 2 Y2 antibodies, Ipg antibody was incubated with Ipg peptide: 1.67pl of antibody (at 0.6mg/ml) was added to 2.5pl of peptide (at 0.4mg/ml) and made up to 333pl with NHS, to give a 1:200 solution of P2Y antibody and incubated at 4°C overnight. The antibody/peptide solutions were then agitated and

incubated for a further 1 hour at 4°C. The mixture was passed through a syringe filter (0.2pm) and then centrifuged for 5 minutes at 13,000rpm|(Biofuge Fresco Centrifuge, Heraeus) and the supernatant used for immunohistochemistry.

81 Double-labelling techniques

Double-labelling with P2X or P2Y receptor antibodies and either PCNA, cytokeratin KIO, or involucrin. Proliferating cell nuclear antigen (PCNA) (Miyagawa et al. 1989) is a marker for proliferation in normal human kératinocytes. Cytokeratin KIO (Eckert et al. 1997) and involucrin (Eckert et al. 1997) are markers for keratinocyte differentiation. Each of these antibodies was raised in mouse. Sections were fixed for 2 minutes in 4% formaldehyde in O.IM phosphate buffer, containing 0.2% of a saturated solution of picric acid (pH 7.4). After a wash step, sections were pre-incubated for 20 minutes at room temperature in 10% NHS in PBS-merthiolate, followed by incubation with the P2X or P2Y receptor antibody overnight, diluted at 1:100 or 1:200 as before. Biotinylated donkey anti-rabbit IgG antibody, diluted 1:500 in 1% NHS in PBS, was then applied for 1 hour followed by Streptavidin Texas red (Amersham International pic., UK), diluted 1:200 in PBS- merthiolate for 1 hour at room temperature. P2X or P2Y receptor immunostaining was observed as a red colour. Sections were pre-incubated for 30 minutes with 10% normal goat serum (NOS) diluted in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, Poole, UK), and then incubated for 2 hours at room temperature with one of the following antibodies: PCNA antibody (monoclonal anti-proliferating cell nuclear antigen, clone PC 10, raised in mouse ascites fluid; Sigma) diluted 1:1000; mouse anti human cytokeratin KIO (BioGenex, San Ramon, CA, USA) diluted 1:50; mouse monoclonal anti-involucrin (Sigma) diluted 1:50. After a wash step, the directly labelled secondary antibody goat anti-mouse FITC (Nordic) was applied at a dilution of 1:200 for 1 hour and then sections were washed and mounted in Citifluor (Citifluor Ltd.).

Double-labelling with P2X or P2Y receptor antibodies and either Ki-67 antigen or anti-human caspase-3 Ki-67 antigen is a marker for cell proliferation in normal human kératinocytes (Gerdes et al. 1991; Tucci et al. 1998). Active caspase-3 is part of the apoptotic machinery of the cell and is expressed in terminally differentiating kératinocytes (Weil et al. 1999). Rabbit anti-human Ki-67 antigen (DAKO, Denmark) and rabbit anti-human active caspase-3 (Abeam, UK) were used. Sections were fixed for 2 minutes in 4% formaldehyde in O.IM phosphate buffer, containing 0.2% of a saturated solution of picric acid (pH 7.4). Sections were immersed for 10 minutes in 50% methanol containing 0.4% hydrogen peroxide. After a wash step, sections were pre-incubated for 20 minutes in 10% normal horse serum

82 (NHS) in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, Poole, UK), followed by incubation with either P2X or P2Y receptor antibodies diluted to 1:100 or 1:200 in antibody diluent (10% NHS in PBS + 2.5% NaCl) at 4°C overnight. Subsequently, the sections were incubated with biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Lab) diluted to 1:500 in 1% NHS in PBS for 1 hour, followed by ExtrAvidin peroxidase conjugate (Sigma) diluted to 1:1500 in PBS for 1 hour, tyramide amplification for 8 minutes (Tyramide Amplification Kit, NEN Life Science Products, Boston, MA) and then Streptavidin Texas red (Amersham International pic., UK), diluted 1:200 in PBS-merthiolate for 10 minutes. Sections were washed three times in PBS after each of the above steps. Sections were pre-incubated for 20 minutes in 10% NOS and then incubated at room temperature for 2 hours with one of the following antibodies: rabbit anti-human Ki-67 antigen (DAKO, Denmark) 1:50 or rabbit anti-human active caspase-3 (Abeam, UK). Sections were then washed and incubated with the directly labelled secondary antibody, Oregon-green-labelled goat- anti-rabbit IgG (Jackson ImmunoResearch Lab), diluted 1:100 for 45 minutes. Sections were then washed and mounted in Citifluor (Citifluor Ltd.).

Double-labelling with PlXy receptor antibodies and TdT-mediated dUTP nick end labelling (TUNEL) TUNEL labelling was performed using a kit (Boehringer Mannheim, Germany). TUNEL identifies cells undergoing apoptosis by labelling nuclear DNA fragments that have been cleaved during apoptosis (Gavrieli et al. 1992). After overnight incubation with P2X? receptor antibody diluted to 1:200 as before, sections were washed in PBS and then incubated with the TUNEL reaction mixture for 1 hour at 37°C. As a negative control, sections were incubated with the TUNEL Label solution only. After further washes in PBS, sections were incubated for 1 hour with biotinylated donkey anti-rabbit antibody at a concentration of 1:500. Sections were washed in PBS and then incubated for 1 hour with Streptavidin Texas red (Amersham International pic., UK) at a concentration of 1:200. Sections were washed in PBS and mounted in Citifluor (Citifluor Ltd.). Photography The results were photographed using a Zeiss Axioplan, high definition light microscope (Oberkochen, Germany) mounted with a Leica DC 200 digital camera (Heerbrugg, Switzerland).

83 Human primary cultures of kératinocytes in low calcium medium Human keratinocyte primary cultures were grown from post-operatively redundant skin of consenting patients under age 30, who had undergone prominent ear correction, breast reduction and abdominoplasty. Cultures were grown in the presence of irradiated 3T3 cells which were used as feeder layers (Rheinwald, 1980). Kératinocytes at passage 0 were disaggregated via trypsin at approximately 70% confluency and seeded (passage 1) into low calcium keratinocyte medium. Low calcium medium was made up with 200mls of MCDB 153 medium, lOmls of chelated fetal calf serum, 2mls of RM+ medium, 2mls of penicillin/streptomycin mixture, 2mls of L- glutamine and filter sterilized (0.2pm) before use. The cells were media-changed at 24 hours after passage to remove the non-adherent, dead kératinocytes and feeder cells that remained floating in the medium. At 48 hours after passage, the cells were disaggregated with trypsin/EDTA and seeded into 96 well plates at a density of 4 xlO^ cells/well in low calcium medium for use in the proliferation assay. Proliferation Assay Twenty-four hours after seeding into 96 well plates, medium was gently aspirated from culture wells, and concentration ranges of PI and P2 receptor subtype agonists and antagonists were applied to the kératinocytes, diluted in low calcium keratinocyte medium. These included: the PI receptor agonist, adenosine (Sigma); adenosine 5’-triphosphate (ATP; Sigma); 8 -(p-sulfophenyl)theophylline ( 8 /?SPT;

Sigma), the A 1/A2 adenosine receptor antagonist (Bruns et al. 1980, 1986); dipyridamole (Sigma), the adenosine transport blocking agent (Maguire and Satchell

1979); uridine 5’-triphosphate (UTP; Sigma), P 2Y2 receptor agonist (von Kiigelgen and Wetter 2000); adenosine 5’-0-(3-thiotriphosphate) (ATPyS; Sigma), P2Xs receptor agonist (Haines et al. 1999b; Khakh et al. 2000; Lambrecht 2000) 2’-& 3’-0-(4- Benzoyl-benzoyl) adenosine 5’-triphosphate (BzATP; Sigma), P2X? receptor agonist (Ralevic and Bumstock 1998; Khakh et al. 2001), 2-Methylthioadenosine 5’- diphosphate (2MeSADP; Sigma), P2Yi receptor agonist (von Kiigelgen and Wetter 2000) and Suramin (Sigma), a non-selective P2X and P2Y receptor antagonist (Ralevic and Bumstock 1998). The pH of the dmg solutions made up in culture medium was 7.0. Changes in cell number were quantified via a colorimetric assay using crystal violet (Gillies et al. 1986; Kueng et al. 1989) and read using a spectrophotometric plate reader (Model 550, Microplate Manager® 4.0 Bio-Rad Laboratories, Inc) at 0, 24, 48 and 72 hours after addition of dmgs. For the colorimetric assay, a solution of 0.5g crystal violet, 0.85g NaCl, 5mis 10% formal saline, 50mls absolute ethanol, 45mls

84 distilled H2O was used. Medium was gently aspirated from wells of a 96 well plate and lOOp-1 of the colorimetric assay mixture was added to each well and incubated at room temperature for 10 minutes. This mixture allowed simultaneous fixation of cells and penetration of the crystal violet dye into the cells. After washing three times in PBS, 33% acetic acid was used to elute colour from cells and optical density was read at 595nm using the spectrophotometric plate reader. The changes in cell number picked out by the crystal violet assay were validated at least once for each drug set by doing actual cell counts with a haemocytometer. To confirm that the optical density of the wells correlated with cell numbers, a control assay was performed for each experiment, where known numbers of cells were seeded in ascending seeding densities and the plate read as soon as cells had attached. Cell number versus optical density was plotted. The value of the trend line was always >0.98. Statistical analysis Each proliferation assay experiment was repeated an average of eight times, each with triplicate samples. Data analysis was performed using Microsoft Excel 97 and GraphPad Prism 3.0 software. One-way analysis of variance (ANOVA) and Bonferroni’s multiple comparison test were carried out between groups. Subcutaneous injection experiment Skin was harvested from the back of adult male Wistar rats and 0.1ml of ATP, ATPyS, BzATP, UTP or 2MeSADP was injected subcutaneously into the dermis, using a 1ml syringe and a 270 needle, at concentrations from l-500pM. Controls had 0.1ml of saline injected in the same way. The area of the injection site was marked on the skin with a marker pen and the solution infiltrated subcutaneously to a diameter of about 0.5cm around the injection site. The skin was then placed in Hank’s balanced salt solution and kept at 4°C for one hour, after which the tissue was frozen in isopentane pre-cooled in liquid nitrogen. Blocks were sectioned at 10pm on a cryostat (Reichert Jung CM1800) until the marked area indicating the injection site was visible. Sections were collected on gelatin-coated slides and air-dried at room temperature. The injection site was indicated by blue ink on unstained slides. The slides were stained with haematoxylin and eosin and compared to unstained serial sections to reveal the injection site. From previous work (Terenghi et al. 1994), it was established that n =6 was the minimum size per group to avoid inter-animal and inter-sample variation influencing the outcome of the results. This group size ensured that the statistical significance was at the 5% level, with an 80% statistical power. Each drug set was therefore repeated in six different animals.

85 RESULTS

P2Xs and P2X? receptors are expressed in human skin

P2X5 and P2X7 receptor immunoreactivity was observed in the epidermal kératinocytes of all human skin samples. P2X$ receptor staining was restricted to the first two viable layers of the epidermis, with very few stained cells in the stratum granulosum (Fig 2.1a). No P2Xs receptor staining was found in the stratum comeum. Higher magnification revealed differences in staining for this receptor between the stratum basale and the stratum spinosum in both intensity and subcellular distribution, with staining of the stratum basale appearing slightly less intense than that of the stratum spinosum (Fig 2.1b). In addition, staining within the stratum spinosum was mainly in the cytoplasm and possibly the plasma membrane, but not in the nucleus, whereas in the stratum basale the majority of staining appeared to be in the plasma membrane. Furthermore, this staining of the stratum basale cell membranes appeared to be polarised, being mainly concentrated on the basal aspects of the cells that were associated with the basement membrane. P2X? receptor immunoreactivity was solely associated with cells and cell fragments within the stratum comeum (Fig 2.1c, 2.1d), and were often seen to be nuclear or peri-nuclear. Some differences were noted in the pattern and intensity of staining between the different skin areas. In lower leg skin, there was strong immunostaining for P 2X5 receptors in all the cells of the stratum basale and stratum spinosum (Fig 2.1a, 2.1b). In breast (Fig 2.2a) and abdominal skin (Fig 2.2c), P2Xs immunoreactivity was similar to that in the lower leg with strong P2Xg receptor staining within the stratum basale and spinosum. However, in these tissues, the stratum granulosum showed more intense

P2X5 immunoreactivity and almost all of the cells in the stratum granulosum stained positive for the P2X$ receptor antibody. In lower leg skin (Fig 2.1c, 2.1d), comeocytes and cell fragments within the stratum comeum stained positively for P2X? receptors. The staining was of high intensity compared to that seen in the other areas examined. For example, in breast skin, which is thin, the staining for P2X? receptors was at the junction between the stratum granulosum and the stratum comeum in an almost continuous thin line (Fig 2.2b) There was little staining of the stratum comeum in breast skin compared to that seen in the lower leg, abdomen and thigh skin. In abdominal skin (Fig 2.2d), the P2X? receptor staining was in an almost continuous line, and staining of the stratum comeum was patchy. In thigh skin, membrane fragments within the stratum comeum stained

86 positively for P2X? receptors, which were also sometimes expressed on the outer surface of the stratum comeum (Fig 2.2e). In leg skin, the epidermis was thicker and the zones of the epidermis were better defined than in other regions of the skin, therefore this was used for further studies.

P2Yi and P 2 Y2 receptors are expressed in human skin

P2Yi (Fig 2.3a) and P2Y2 receptors (Fig 2.3b) were expressed in normal human skin. P2Yi receptors were found in the stratum basale of the epidermis. P 2Y2 receptors were also found in the stratum basale of the epidermis, with some expression in the stratum spinosum.

Ceils positive for P2Yi and P 2 Y2 receptors also express markers for cellular proliferation. Neonatal skin was dual stained for Ki-67 and P2Yi receptors (Fig 2.4a) and

PCNA and P2 Y2 receptors (Fig 2.4b). The proliferation markers identified a proliferating subpopulation of basal and parabasal kératinocytes. Cells positive for these two markers were also positive for P2Yi and P 2 Y2 receptors. Cells positive for P2Xs receptors also express markers for keratinocyte differentiation. Double-labelling of P2Xs receptors with cytokeratin KIO (Fig 2.4c) or involucrin (Fig 2.4d) showed that P2Xg receptors were expressed in differentiating kératinocytes within the epidermis. Cytokeratin KIO, an early marker of keratinocyte differentiation was found in most suprabasal kératinocytes. The stratum basale stained only for P 2X5 receptors, indicating that no differentiation was taking place in these cells. The co-localisation of P2Xs receptors and cytokeratin KIO appeared mainly in the cytoplasm of differentiating cells within the stratum spinosum and partly in the stratum granulosum. Involucrin, a late marker of keratinocyte differentiation was expressed from the upper stratum spinosum up to the stratum comeum. There was co-localisation of P 2X5 receptors with involucrin within the cytoplasm of cells within the upper stratum spinosum, with a few cells double-labelling within the stratum granulosum. Double-labelling of P2X? receptors with markers for apoptosis in human skin show co-localisation. The cell nuclei in the uppermost level of the stratum granulosum stained strongly positive for TUNEL (Fig 2.4e) and caspase-3 (Fig 2.4f). Double-staining of the

P2X7 receptor and TUNEL showed that there was some overlap of staining, but that TUNEL largely stained the cell nuclei of living cells undergoing apoptosis in the

87 uppermost layer of the stratum granulosum, while P2X? immunostaining also stained the dead cells in the stratum comeum above. The negative control for TUNEL showed no reaction. Double labelling of P2X? receptors with anti-caspase-3 showed co localisation within the stratum comeum (Fig 2.4f). Control immunostaining experiments Both the omission of the primary antibody and preabsorption with corresponding peptides were performed as controls. With immunofluorescence staining, there was some non-specific immunostaining of nuclei within the stratum spinosum and granulosum when the primary antibody was omitted, but there was no immunoreaction when the secondary and tertiary antibodies were omitted. The immunoreaction was abolished after preabsorption of the P2Xs (Fig 2.2f) or P2X? receptor antibodies (Fig

2 .2 g) with the corresponding peptides, confirming the specificity of the immunoreaction. The immunoreaction was significantly reduced after preabsorption of the P2Yi (Fig 2.3c) or P 2Y2 antibody (Fig 2.3d) with the corresponding peptide, with some non-specific staining of nuclei within the stratum spinosum and granulosum, confirming the specificity of the findings. Western blotting could have helped to further confirm the specificity of the antibody. Functional studies of primary keratinocyte cultures show that purinergic receptor agonists can alter cell number. Readings were taken of changes in optical density of cells grown in 96 well plates at 0, 24, 48 and 72 hours after drug application. The peak effect of the drugs was seen at 48 hours after application, after which the effect decreased. At 48 hours after application of drugs to primary cultures of human kératinocytes, ATP had a more potent effect than adenosine (Fig 2.5a). The effect of adenosine was not blocked by the adenosine (A 1/A2) receptor antagonist, 8 -(p- sulfophenyl)theophylline (Fig 2.5b). The adenosine transport blocking agent, dipyridamole, had an inhibitory effect of its own, and did not potentiate the effect of adenosine (Fig 2.5c). The effect of ATP was not significantly reduced by 8 -(p- sulfophenyl)theophylline (Fig 2.6a), which meant that the effects of ATP were not mediated via ATP breaking down to adenosine and activating extracellular adenosine receptors. There was no potentiation of the ATP response by dipyridamole (Fig 2.6b) ATP (1-lOpM), UTP (lOOpM) (P<0.001) (Fig 2.7) and 2MeSADP (500pM) (P<0.05) (Fig 2.8) caused an increase in keratinocyte cell number. Whereas ATP (lOOpM) (P<0.001), ATPyS (100-500pM) (P<0.001) (Fig 2.7) and BzATP (100-500pM) (P<0.001) (Fig 2.9) caused a significant decrease in cell number. Suramin (Fig 2.10)

88 caused a significant block in the effect of high dose ATP at lOOfiM (?<0.01) and 1000^M(P<0.001). Direct subcutaneous injection of purinergic receptor agonists results in changes in the morphology of the rat epidermis. No change in the morphology or thickness of the epidermis was seen when 0.1 mis saline was injected subcutaneously into control skin (Fig 2.11a, 2.11b). Subcutaneous injection of either O.lmls lOpM ATP (Fig 2.11c, 2.11d), or lOOpM UTP

(Fig 2.11e) resulted in a thickening of the rat epidermis compared to control skin, in 6 out of 6 rats studied. Subcutaneous injection of SOOpM 2MeSADP (Fig 2.11f) caused an increase in thickness of the epidermis in 3 out of 6 rats, with 3 showing no change. lOpM ATP caused a three-fold increase in the thickness of the epidermis (Fig 2.1 Id), whereas IGOpM UTP caused a two-fold increase in thickness (Fig 2.11g). Both ATP and UTP increased the number of viable cell layers, with a UTP causing a smaller increase in the stratum granulosum and stratum comeum. 2MeSADP'also caused an increase in the number of viable cell layers (Fig 2.1 Ih). Further work would be needed to confirm whether the change in thickness was due to hypertrophy or hyperplasia. Subcutaneous injection of 300pM ATPyS (Fig 2.11i, 2.11k) caused a slight

decrease in thickness of the epidermis in 3 out of 6 rats, with the remaining 3 preparations showing no change compared to control. Subcutaneous injection of 300pM BzATP (Fig 2.1 Ij) caused a decrease in thickness of the viable cell layers of the

epidermis in 4 out of 6 rats studied (in 2 out of 6 there was no change). The thickness of the epidermis was almost halved and the cells of the stratum granulosum cells were flattened and thinner than in control skin (Fig 2.111). The concentration of 300pM was chosen for ATPyS and BzATP because this represented the midpoint of the range of effective concentrations used within human primary keratinocyte cultures.

89 DISCUSSION

Previous work has shown that adenosine and ATP have an anti-proliferative

effect on cultured human kératinocytes (Cook et al. 1995) and that adenosine À 2b receptor mRNA is present in human kératinocytes (Brown et al. 2000). In this study, ATP had a more potent effect than adenosine. The anti-proliferative effect of adenosine

was not blocked by the A 1/A2 receptor antagonist, 8 -(p-sulfophenyl)theophylline, confirming the findings in a previous study which used theophylline (Brown et al. 2000). The adenosine transport blocking agent, dipyridamole had an inhibitory effect of its own and did not potentiate the effect of adenosine. This suggested that extracellular adenosine receptors were not being activated in this system. The effects of adenosine might be mediated via an intracellular site of action as suggested by previous work

(Brown et al. 2000). In the present study, the response to ATP was not blocked by 8 -(p- sulfophenyl)theophylline, which meant that the effect of ATP was not due to breakdown of ATP to adenosine and activation of extracellular adenosine receptors. Dipyridamole did not potentiate the ATP response, so, blocking adenosine transport into the cell, and thereby increasing local extracellular adenosine concentrations did not have any effect. This suggests that the main trophic agent in human keratinocyte cultures is ATP, working via P2 receptors, while adenosine has a minor effect, which is largely obscured by the ATP response. This study focused on further investigation of the expression and functional roles of P2 purinergic receptor subtypes in the normal epidermis.

In this study, the first direct evidence for the expression of P2Yi and P 2 Y2 receptors in human kératinocytes was obtained using immunohistochemistry and functional studies in vitro. P2Yi and P 2 Y2 receptors show a strong immuno-positive signal in the basal layer of the epidermis. Some basal cells are stem cells, others are transit amplifying cells (which can undergo a variable number of rounds of division before becoming post-mitotic) and some are post-mitotic cells that are poised to move into the suprabasal layers. Double-labelling of P2Yi and P 2 Y2 receptors and keratinocyte proliferation markers Ki-67 and PCNA was performed. This confirmed the presence of P2Yi and P 2 Y2 receptors in proliferating cells. This study has shown that the P2Yi and P2 Y2 receptor selective agonists, 2MeSADP and UTP respectively, can cause an increase in human keratinocyte number in vitro. Direct subcutaneous injection of UTP into rat skin also causes a two-fold increase in thickness of the epidermis. This suggests that these receptors are involved in keratinocyte proliferation. Since the effect of 2MeSADP is weaker than that of UTP, it also suggests that P 2 Y2 receptors may have

90 a more significant role in this process. Previous work has localised P 2 Y2 receptor mRNA in human epidermal basal cells via in situ hybridisation (Dixon et al. 1999), in human retinal pigment epithelial cells (Sullivan et al. 1997), equine epithelial cells (Wilson et al. 1998), and frog skin epithelium (Brodin and Nielsen 2000). UTP has also been shown to cause proliferation in kératinocytes (Dixon et al. 1999) and HaCaT cells (Lee et al. 2001). In this study, the distribution of P2X$ receptors within human epidermis has been shown for the first time. These receptors are also expressed in the stratum basale, but are expressed more strongly in the stratum spinosum and variably into the stratum granulosum. The position of the keratinocyte within the epidermis correlates with its state of differentiation. Differentiation of the basal keratinocyte results in a permanent loss of growth potential and the subsequent sequential expression of differentiation markers. The first markers expressed are the two suprabasal keratins K1 and KIO (Schweizer and Winter 1983; Eichner et al. 1986) followed by other proteins such as involucrin (Watt 1983), which is detected in the late stratum spinosum and in the stratum granulosum. Double-labelling of P2Xs receptors with either cytokeratin KIO or involucrin was performed to demonstrate that P2Xs receptors are expressed on differentiating kératinocytes. There is a striking overlap of expression of P2Xg receptors with keratin KIO, an early differentiation marker but much less overlap yvith involucrin, which is expressed later. P 2Xs receptors may be more likely to be involved in keratinocyte differentiation because ATPyS, a potent P 2X5 receptor agonist, causes a significant decrease in cell number in culture. This may be because kératinocytes are withdrawing from the cell cycle and differentiating. ATPyS may cause premature differentiation of the upper layers of the epidermis in rat skin, reducing the number of viable cell layers after subcutaneous injection. There is also evidence from other tissues regarding the role of P 2X5 receptors. P2Xg receptors are sequentially expressed during development in rat (Ryten et al. 2001). P2Xs receptors have been implicated in the regulation of osteoblastic differentiation and proliferation (Hoebertz et al. 2000) and in triggering the differentiation of skeletal muscle satellite cells (Ryten et al. 2002). The co-localisation of P2X? receptors with two different apoptosis markers, TUNEL and anti-caspase-3 in the stratum comeum is of particular interest. The P2X? receptor is unlike other P2X receptors because it is a bifunctional molecule, that can be triggered to act as a channel permeable to small cations, or on prolonged stimulation form a cytolytic pore permeable to large hydrophilic molecules up to 900 Da (Surprenant et al. 1996). The opening of this pore results in the increase in intracellular

91 cytosolic free calcium ions. The P2X? receptor is involved in the release of IL-ip (Ferrari et al. 1997a) and the induction of cell death (Zheng et al. 1991; Ferrari et al. 1996). P2Xv receptors are also found on dendritic cells, macrophages and microglial cells, where extracellular ATP can trigger apoptosis via these receptors and there is increasing evidence that this process is dependent on the caspase signalling cascade (Coutinho-Silva et al. 1999; Ferrari et al. 1999). Caspase-3 is considered an executioner caspase, which once activated will cleave a number of cellular substrates whose limited proteolysis is definitive of apoptosis (Nicholson and Thomberry 1997). Caspase-3 is expressed in terminally differentiating kératinocytes (Weil et al. 1999). During apoptosis, the nucleus condenses and DNA is fragmented by endonucleases, which can be detected by TdT-mediated dUTP nick end labelling (TUNEL). TUNEL-positive kératinocytes have been found in the upper regions of the granular layer of the epidermis, before comification (Polakowska et al. 1994; Tamada et al. 1994; Gandarillas et al. 1999). These markers suggest that part of the apoptotic machinery of the cell is activated during keratinocyte terminal differentiation and that this activation may be required for the normal loss of the nucleus in comeocyte formation. Our study suggests that P2X? receptors are also likely to be part of that machinery. There has been much debate about whether terminal differentiation of kératinocytes is a specialised form of apoptosis (Haake and Polakowska 1993; Polakowska et al. 1994; Maruoka et al. 1997). Classical apoptosis and terminal differentiation differ. Terminal differentiation of kératinocytes is a form of genetically programmed cell death (Paus et al. 1993). It generates keratin-packed comeocytes and a functionally important epidermal barrier. Terminal differentiation occurs synchronously in all kératinocytes in the suprabasal layers whereas apoptosis occurs in individual cells (Kerr et al. 1972). During apoptosis, DNA is cleaved by endonucleases and the nucleus condenses and fragments. There is blebbing of the plasma membrane and the cell breaks up into discrete, membrane bound fragments, while the cytoplasmic organelles and plasma membrane retain their integrity until the final stages. Neighbouring cells or macrophages rapidly phagocytose the dead cells. Kératinocytes do not undergo membrane blebbing and fragmentation during terminal differentiation and are not phagocytosed by neighbouring cells but are sloughed from the surface of the tissue. The time required for execution of the differentiation pathway takes days as opposed to the minutes required for the final stages of apoptosis. Finally, the protein components required for apoptosis are expressed in all nucleated mammalian cells (Jacobson et al.

92 1997), but terminal differentiation involves expression of a large number of keratinocyte-specific genes (Fuchs and Byrne 1994). There are clear similarities between apoptosis and epidermal terminal differentiation. Both are metabolically active processes that provoke a series of dramatic cellular changes and ultimately lead to cell death. Both are tightly controlled by interaction with the extracellular matrix via integrins (Gandarillas 2000), and when this interaction is disrupted, kératinocytes undergo terminal differentiation (Green 1977) and endothelial cells and some epithelial cells undergo apoptosis (Frisch and Ruoslahti 1997). Common features are also activation of transglutaminases (Fesus et al. 1991; Nagata 1997), essential for the production of the keratinocyte comified envelope and the loss of the nucleus (Rice et al. 1994). Also, c-Myc induces apoptosis and also stimulates keratinocyte differentiation (Gandarillas and Watt 1997). This study has shown that P2Xv receptors on kératinocytes are functional, cell numbers are significantly reduced when stimulated with BzATP in culture, and subcutaneous injection of BzATP appears to cause premature differentiation or death of the upper layers of the epidermis in rat, leaving behind one to two viable cell layers. If P2Xv receptors are involved in the terminal differentiation programme, which ends in ‘cell death’ and formation of the keratinised anucleate comeocyte, then cells grown in culture may also die by this mechanism. ATP in high doses has a similar effect to that of BzATP in culture and it is likely that ATP is stimulating P2X? receptors on kératinocytes. This has been demonstrated in other cell systems (North and Surprenant

2000 ) and this study has shown that this effect can be significantly blocked by suramin in culture. Suramin is a non-specific antagonist of P2X and P2Y receptors. In summary, P2 purinergic receptor agonists alter keratinocyte cell numbers via several mechanisms, namely UTP and low concentrations of ATP cause an increase in cell number, probably via a direct proliferative effect on basal cells via P 2 Y2 receptors.

2MeSADP also leads to an increase in cell number, probably via P2Y 1 receptors in the basal layer. ATPyS results in a decrease in cell number because cells are lost from the cell cycle by being made to differentiate via activation of P2Xs receptors. BzATP and high concentrations of ATP decrease cell numbers via a direct effect on P2X? receptors, which are known to be involved in mediating apoptosis and are likely to be part of the machinery of end stage terminal differentiation of kératinocytes.

93 FIGURE LEGENDS

Figure 2.1: Human epidermal kératinocytes in frozen sections of leg skin expressed

P2Xs and P 2 X7 receptors

(a) P2X5 receptor immunostaining was present in the stratum basale and stratum spinosum. Scale bar 40pm. (b) High power view of (a): P2Xs receptor immunostaining was present in the stratum spinosum (SS) and to a lesser extent in the stratum basale (SB). Only a few cells in the stratum granulosum (SG) had positive staining, which was of much lower intensity. There was no staining in the stratum comeum (SC). Scale bar 15pm. (c) P2X? receptor immunostaining was present in whole cell remnants and cell fragments within the stratum comeum. Scale bar 40pm. (d) P2X? receptor immunostaining- high power view. Staining was often seen to be nuclear or peri nuclear (arrows). Scale bar 15pm.

94 € ' «

95 Figure 2.2: Human epidermal kératinocytes from different regions of the body also expressed P2Xs and P2X? receptors in a similar fashion to that seen in leg skin in Fig 2.1.

(a) Breast skin: P2X5 receptor immunostaining was intense in the stratum basale, spinosum and granulosum. Scale bar 15pm. (b) Breast skin: P2X? receptor immunostaining was present at the junction of the stratum granulosum and the stratum comeum. There was little staining within the stratum comeum. Scale bar 15pm. (c) Abdomen skin: P2Xs receptor immunofluorescence staining (white) was intense in the stratum basale, spinosum and granulosum. Scale bar 30pm. (d) Abdomen skin: P2X? receptor immunofluorescence staining (white) was seen at the junction between the stratum granulosum and the stratum comeum. There was patchy staining of the stratum comeum. Scale bar 30pm. (e) Thigh skin: Immunopositive areas for P2X? receptors

were found both within the stratum comeum and on the outer surface. Scale bar 15pm. (f) Leg skin: Immunoreaction was abolished after absorption of the P2X$ antibody with an excess of the corresponding peptide used for inununisation. Scale bar 60pm. (g) Leg skin: Immunoreaction was significantly reduced after absorption of the P2Xv antibody with an excess of P2X? peptide. Scale bar 60pm.

96 a

------

97 Figure 2.3: Human epidermal kératinocytes from leg skin expressed P2Yi and

P2 Y2 receptors. (a) P2Yi receptors were found in the basal layer of the epidermis. Scale bar 20pm. (b)

P2 Y2 receptors were found in the basal layer of the epidermis, with some expression in the stratum spinosum. Scale bar 20pm. (c) The immunoreaction was significantly reduced after absorption of the P2Yi antibody with P2Yi peptide. Scale bar 60pm. (d)

The immunoreaction was significantly reduced after absorption of the P 2 Y2 antibody with P2 Y2 peptide. Scale bar 60pm.

98 99 Figure 2.4: Double-labelling of P2Yi and P 2 Y2 receptors with markers of proliferation showed co-localisation within a suhpopulation of basal and parabasal kératinocytes. Double-labelling of P2Xs receptors with markers of differentiated kératinocytes showed co-localisation within the stratum spinosum, and double labelling of P 2 X7 receptors with markers of apoptosis in human leg skin showed co-localisation within the stratum corneum. (a) Ki-67 immuno-labelling (a marker for proliferation) stained the nuclei (green) of a subpopulation of kératinocytes in the basal and parabasal layers of the epidermis. P 2 Y% receptor immunostaining (red) was found in the basal layer on cells also staining for Ki67. Scale bar 30pm. (b) PCNA immuno-labelling (a marker for proliferation) stained the nuclei (green) of a subpopulation of kératinocytes. These nuclei were often distributed in clusters and found in the basal and parabasal layers of the epidermis. P 2Y2 receptor immunostaining (red) was also expressed in basal and parabasal epidermal cells. Scale bar 30pm. (c) P2Xs receptor immunostaining (red) showed overlap (yellow) with cytokeratin KIO (green), an early marker of keratinocyte differentiation. P2Xs receptors were present in the basal layer of the epidermis up to the mid-granular layer. Cytokeratin KIO was distributed in most suprabasal kératinocytes. The basal layer stained only for P2Xs receptors, indicating that no differentiation was taking place in these cells. The co-localisation of P2Xs receptors and cytokeratin KIO appeared mainly in the cytoplasm of differentiating cells within the stratum spinosum and partly in the stratum granulosum. Note that the stratum corneum also stained for cytokeratin KIO, which labelled differentiated kératinocytes, even in dying cells. Scale bar 30pm. (d) P2Xs receptor immunostaining (red) showed overlap (yellow) with involucrin (green).

P2X5 receptors were present in the basal layer of the epidermis up to the mid-granular layer. Note that the pattern of staining with involucrin was similar to that seen with cytokeratin KIO, except that cells from the stratum basale up to the mid-stratum spinosum were not labelled with involucrin, which is a late marker of keratinocyte differentiation. Scale bar 30pm. (e) TUNEL (green) labelled the nuclei of cells at the uppermost level of the stratum granulosum and P2X? antibody (red) mainly stained cell fragments within the stratum corneum. Scale bar 15pm. (f) Anti-caspase-3 (green) co localised with areas of P2X? receptor immunostaining (red) both at the junction of the stratum granulosum and within the stratum corneum. Areas of co-localisation were yellow. Note that the differentiating kératinocytes in the upper stratum granulosum were also positive for anti-caspase-3. Scale bar 15pm.

100 101 ^ ATP R S Adenosine I! Il

< / / / / / / ' ''/yy

drug concentration /jiM

Smni A d enosine dZIaOtiM BpSPT+Z- II ” A denosine □ i II ° ■ = £■ II» T f t ... 1 s« T

drug concentration / pM

]30pM Dipyridamole +/- II A denosine

T T l T / y

drug concentration ZpM

Figure 2.5: At 48 hours after application to primary keratinocyte cultures: a) The effect of 100-5000pM ATP was significantly (P<0.001) more potent than that of the same dose of adenosine, b) 30pM 8 -(p-sulfophenyl)theophylline ( 8 pSPT) caused no significant block of the effect of adenosine, c) 30pM Dipyridamole (Dip) had no significant effect on the response to adenosine. Results represent the mean of up to 7 experiments. Error bars represent mean +/-SEM. (*P<0.01, **P<0.001 compared to control)

102 50-

IHATP

2 5 - IZZ] 30^M SpSPT -t-/- ATP l î ^o "O V

IIC >, - 2 5 - = n "1 - 3 0) OT - 5 0 - g>g ™ E o - 7 5 -

-100 / / / / / / y / y /

drug concentration /^M

25-1 H ! ATP

1 130^M Dipyridamole +/- A T P

0 - î ï

II - 2 5 - I? £5-

8 ? - 5 0 - I s 5

«H E

u - 7 5 -

- 1 0 0 - cP" y _lp y

drug concentration /pM

Figure 2.6: At 48 hours after application of ATP to primary human keratinocyte cultures with either a) 30pM 8 -(p-sulfophenyl)theophylline ( 8 pSPT) or b) 30pM Dipyridamole (Dip), no significant effect on the response to ATP was observed. Results represent the mean of up to 7 experiments. Error bars represent mean 4-/-SEM. (*P<0.01, **?<0.001 compared to control).

103 IllU TP 40n H ATP 30- X CZiATPyS

20 -

10-

I 0 - 8 -10-

•20 -

30- g -g E g 40-

50-

60-

70-

80 / /././././ y ./

drug concentration /[iM

Figure 2.7: Al 48 hours after application, ATP (1-1 OpM) and UTP (lOOpM) caused an increase in cell number of primary human keratinocyte cultures, whereas ATPyS (100-500pM) and ATP (lOOpM) caused a significant decrease.

Results represent the mean of 8 experiments. *P<0.001 compared to control. Error bars represent mean 4-/-SEM.

104 control

concentration 2M58ADP/[iM

Figure 2.8: At 48 hours after application, 2MeSADP (SOOpM) caused a significant increase in cell number of primary human keratinocyte cultures.

Results represent the mean of 8 experiments. *P<0.05 compared to control. Error bars represent mean +/-SEM.

105 10-

0 - il o o

il - 20 -

8% .5 3 0) (0 -30 * g . s (0 c

-40-

-50 control 10 100 500

BzATP concentration /^iM

Figure 2.9: At 48 hours after application, BzATP (100-500pM) caused a significant decrease in cell number of primary human keratinocyte cultures. Results represent the mean of 9 experiments. *P<0.001 compared to control. Error bars represent mean -I-/-SEM.

106 20-1 t

" lîO O £ ^ -10 o u | | . 20.

30-

11(ü w -40 g .s (0 c £ -50

-60-

-70 /S

drug concentration /p.M

Figure 2.10: At 48 hours after application of ATP with lOOpM suramin, suramin caused a significant block in the effect of lOOpM ATP (*P<0.01) and lOOOpM ATP (**P<0.001) on cell number of primary human keratinocyte cultures. Results represent the mean of 4 experiments. Error bars represent mean 4-/-SEM.

107 Figure 2.11: Subcutaneous injection of purinergic receptor agonists caused changes in the thickness of the epidermis of rat skin. (a) Low power view of saline injection site (arrow) in control section. Bar represents region of subcutaneous infiltration of saline. Scale bar 375jLim. (b) High power view of (a) taken from the region where saline was injected subcutaneously in control sections. Saline injection did not affect the thickness of the epidermis. Scale bar 20pm. (c) The epidermis was significantly thickened in the area of the injection site (arrow) of lOpM ATP. The bar represents the area of the epidermis affected by low dose ATP, which was much thicker than the surrounding unaffected epidermis. Scale bar 375pm. (d) High power view of (c) taken from the region where lOpM ATP was injected subcutaneously. Note how the epidermis has increased in thickness three-fold compared to control. Scale bar 20pm. (e, f) Low power view of injection site of lOOpM UTP, and SOOpM 2MeSADP respectively, showing that UTP and 2MeSADP caused an increase in thickness of the epidermis at the injection site (arrow). Scale bar 375pm. (g) lOOpM UTP caused a two-fold increase in epidermis thickness compared to control. Scale bar 20pm. (h) 500pM 2MeSADP caused an increase in thickness of epidermis compared to control, (i, j) Low power view of injection site of 300pM ATPyS and 300pM BzATP respectively, showing that the epidermis was reduced in thickness in the area of the injection site (arrow). Scale bar 375pm. (k) 300pM ATPyS reduced the thickness of the epidermis. Scale bar 20pm. (1) 300pM BzATP almost halved the thickness of the epidermis and the morphology of stratum granulosum cells was changed such that cells were much flatter and thinner compared to control. Scale bar 20pm.

108 y/

■ f

k-:rWQ

109 CHAPTER 3 PURINERGIC RECEPTORS ARE PART OF A SIGNALLING SYSTEM FOR PROLIFERATION AND DIFFERENTIATION IN DISTINCT CELL LINEAGES IN HUMAN ANAGEN HAIR FOLLICLES

110 ABSTRACT

We have investigated the expression of P2Xg, P2X?, P2Yi and P 2Y2 receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation (PCNA and Ki-67), keratinocyte differentiation (involucrin) and apoptosis (anti- caspase-3). Using immunohistochemistry, we showed that P2Xs, P2Yi and P 2Y2 receptors were expressed in spatially distinct zones of the anagen hair follicle. P2Yi receptors were found in the outer root sheath and bulb; P2Xg receptors were found in the inner and outer root sheaths and medulla; P 2 Y2 receptors were found in living cells at the edge of the cortex/medulla and P2X? receptors were not expressed. Co localisation experiments suggested different functional roles for these receptors: P2Y% receptors were associated with bulb and outer root sheath keratinocyte proliferation; P2Xs receptors were associated with differentiation of cells of the medulla and inner root sheaths; P 2Y2 receptors were associated with early differentiated cells in the cortex/medulla which contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.

I l l INTRODUCTION

The cells of the lower portion of the hair follicle bulb are undifferentiated matrix cells. These are rapidly dividing cells which give rise to eight different cell lineages (Niemann and Watt 2002). From within outwards, these include the medulla, cortex, and hair cuticle cell lineages which make up the hair shaft; the inner root sheath cuticle, Huxley’s and Henle’s layers which make up the inner root sheath (1RS); the companion layer and the outer root sheath (ORS) (Niemann and Watt 2002). The hair cycle is divided into periods of hair growth (anagen), which are followed by a regression phase (catagen), when the lower part of the hair follicle undergoes programmed cell death (Cotsarelis 1997), and a resting phase (telogen), before onset of a new growth phase. The anagen hair follicle is an attractive system for studying proliferation, and differentiation. There is increasing evidence that purinergic signalling can have long term, trophic effects on these processes (Abbracchio and Bumstock 1998; Bumstock 2002a). Adenosine triphosphate (ATP) is now recognised as an important messenger molecule for cell-cell communication, with ATP binding specifically to purinergic receptors (Bumstock 1997; Ralevic and Bumstock 1998). Purinergic receptors are classified into two groups: PI receptors are selective for adenosine and P2 receptors are selective for adenosine 5’-triphosphate (ATP) and adenosine 5’-diphosphate (ADP), which act as an extracellular signalling molecules (Bumstock 1978). P2 receptors are divided into two main families: P2X receptors which are ligand-gated ion channels, and P2Y receptors which are G-protein coupled, based on molecular structure, transduction mechanisms and pharmacological properties (Abbracchio and Bumstock 1994). Seven subtypes of P2X receptors (Khakh et al. 2001) and seven subtypes of P2Y receptors (King et al. 2000; Communi et al. 2001) are recognised. There is growing evidence that ATP may act as an important local messenger in the epidermis. Purinergic receptors are expressed on rat cutaneous kératinocytes and functional roles in the regulation of proliferation, differentiation and cell death have been proposed (Groschel-Stewart et al. 1999a). In particular, P2Xs receptors are expressed on cells undergoing proliferation and differentiation, while P2X? receptors are associated with keratinised dead cells.

P2 Y2 receptors, found in the basal layer of normal epidermis, are claimed to be involved in keratinocyte proliferation (Dixon et al. 1999). P2Yi receptors are thought to be mitogenic in endothelial cells (Bumstock 2002a). Previous work on adult human interfollicular epidermis and primary keratinocyte cultures has suggested that P2Yi and

P2 Y2 receptors are involved in keratinocyte proliferation, that P2Xg receptors are likely

112 to be involved in keratinocyte differentiation, while P2X? receptors are likely to be part of the machinery of end stage terminal differentiation of kératinocytes (see Chapter 2). This study demonstrates the distribution of P2X and P2Y receptors in human anagen hair follicles for the first time. We propose that these receptors are part of the normal homeostatic mechanisms controlling hair keratinocyte proliferation and differentiation.

ic S' :V 'c

'■113 MATERIALS AND METHODS

Tissues Eight samples of normal hair-bearing human skin were examined immunohistochemically in this study. Ethical Committee Approval was obtained to harvest human skin. Samples of post-operatively redundant skin from otoplasty, pre- auricular skin tags and from the leg were obtained. Tissue was frozen in isopentane pre cooled in liquid nitrogen. Blocks were sectioned at 10pm on a cryostat (Reichert Jung CM1800), collected on gelatin-coated slides and air-dried at room temperature. The slides were stored at -20°C. Antibodies The immunogens used for production of polyclonal P2Xs and P2Xv antibodies were synthetic peptides corresponding to 15 receptor-type-specific amino acids (AA) in the intracellular C-termini of the cloned rat P2X receptors, as previously described (Groschel-Stewart et al. 1999a). P2Xs and P2X? antibodies (provided by Roche Bioscience, Palo Alto, California, USA) were kept frozen at a stock concentration of

Img/ml. Polyclonal anti-P2Yi and P 2 Y2 antibodies were obtained from Alomone Labs

(Jerusalem, Israel), and corresponded to the third extracellular loop of the P2Y 1 (AA

242-258) and P 2Y2 receptor (AA 227-244). Antibodies were kept frozen at a stock concentration of 0.6mg/ml (P2Yi, P 2 Y2). Proliferating cell nuclear antigen (PCNA) is a marker for proliferation in normal adult human kératinocytes (Miyagawa et al. 1989). Involucrin is a marker for keratinocyte differentiation (Eckert et al. 1997). PCNA (monoclonal anti-proliferating cell nuclear antigen, clone PC 10, raised in mouse ascites fluid; Sigma, Poole, UK) and involucrin (Sigma) antibodies were raised in mouse. Ki- 67 antigen is a marker for cell proliferation in normal human kératinocytes (Gerdes et al. 1991; Tucci et al. 1998). Active caspase-3 is part of the apoptotic machinery of the cell and is expressed in terminally differentiating kératinocytes (Weil et al. 1999). Ki-67 (DAKO, Denmark) and active caspase-3 (Abeam, Cambridge, UK) antibodies were both raised in rabbit. Immunohistochemistry For immunostaining of cryostat sections, the avidin-biotin technique was used according to a revised protocol (Llewellyn-Smith et al. 1992, 1993). Air dried sections were fixed for 2 minutes in 4% formaldehyde in O.IM phosphate buffer, containing 0.2% of a saturated solution of picric acid (pH 7.4). Endogenous peroxidase was

114 blocked for 10 minutes with 50% methanol containing 0.4% hydrogen peroxide. Non

specific binding sites were blocked by a 20 minute pre-incubation in 10% normal horse serum (NHS) in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, Poole, UK), followed by incubation with the primary antibodies diluted to 1:100 or 1:200 in antibody diluent (10% NHS in PBS + 2.5% sodium chloride (NaCl) at 4°C overnight. Subsequently, the sections were incubated with biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA, USA) diluted to 1:500 in 1% NHS in PBS for 30 minutes, followed by ExtrAvidin peroxidase conjugate (Sigma) diluted to 1:1000 in PBS for 30 minutes at room temperature. After a wash step, a nickel- diaminobenzidine (DAB) enhancement technique was used to visualise the reaction product. Sections were washed three times with PBS after each of the above steps except for after pre-incubation with 10% NHS. After the last wash, sections were dehydrated twice in isopropanol and mounted with EUKTTT (BDH Laboratory Supplies, Poole, UK). Control experiments were carried out with primary antibodies omitted from the staining procedure or the primary antibodies preabsorbed with the corresponding peptides. For the preabsorption control, 1.5pl of the anti-P2X antibody (at 1 mg/ml) was incubated with 24pl of the respective peptide at 5mg/ml overnight at 4°C. 275pi of NHS was added to give 300pl of a 5pg/ml concentration of the P2X antibodies. For P2Yi and

P2 Y2 antibodies, Ipg antibody was incubated with Ipg peptide: 1.67pl of antibody (at 0.6mg/ml) was added to 2.5pl of peptide (at 0.4mg/ml) and made up to 333pl with NHS, to give a 1:200 solution of P2Y antibody and incubated at 4°C overnight. The antibody/peptide solutions were then agitated and incubated for a further 1 hour at 4°C. The mixture was passed through a syringe filter (0.2pm) and then centrifuged for 5 minutes at 13,000rpm and the supernatant used for immunohistochemistry.

Double-labelling techniques

Double-labelling of P2X or P2Y receptor antibodies with either PCNA or involucrin. Sections were fixed and incubated with P2X or P2Y antibodies overnight as described above. After a wash step, biotinylated donkey anti-rabbit IgG antibody, diluted 1:500 in 1% NHS in PBS, was then applied for 1 hour followed by Streptavidin Texas red (Amersham International pic., UK), diluted 1:200 in PBS-merthiolate for 1 hour at room temperature. Sections were pre-incubated for 30 minutes with 10% normal goat serum (NGS) diluted in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, UK). They were then incubated for 2 hours at room temperature with one of the 115 following antibodies: PCNA antibody (Sigma, UK) diluted 1:1000; mouse monoclonal anti-involucrin (Sigma, UK) diluted 1:50. After a wash step, the directly labelled secondary antibody goat anti-mouse FTTC (Nordic Immunological Laboratories, Tilburg, The Netherlands) was applied at a dilution of 1:200 for 1 hour and then sections were washed and mounted in Citifluor (Citifluor Ltd., Leicester, UK). P2X or P2Y receptor immunostaining appeared red and PCNA and involucrin stained green.

Double-labelling of P2X or P2Yreceptor antibodies with either Ki-67 antigen or anti human caspase-3 Sections were fixed and incubated with P2X or P2Y antibodies overnight, as described above. After washing, sections were incubated with biotinylated donkey anti rabbit IgG (Jackson ImmunoResearch Lab) diluted to 1:500 in 1% NHS in PBS for 1 hour, followed by ExtrAvidin peroxidase conjugate (Sigma, UK) diluted to 1:1500 in

PBS for 1 hour, tyramide amplification for 8 minutes (Tyramide Amplification Kit, NEN Life Science Products, Boston, MA) and then Streptavidin Texas red (Amersham International pic., UK), diluted 1:200 in PBS-merthiolate for 10 minutes. Sections were washed three times in PBS after each of the above steps. Sections were pre-incubated for 20 minutes in 10% NGS and then incubated at room temperature for 2 hours with one of the following antibodies: rabbit anti-human Ki-67 antigen (DAKO, Denmark) 1:50 or rabbit anti-human active caspase-3 (Abeam, UK). Sections were then washed and incubated with the directly labelled secondary antibody, Oregon-green-labelled goat-anti-rabbit IgG (Jackson ImmunoResearch Lab), diluted 1:100 for 45 minutes. Sections were then washed and mounted in Citifluor (Citifluor Ltd., UK). P2X or P2Y receptor immunostaining appeared red; Ki67 and caspase-3 stained green. Photography The results were photographed using a Zeiss Axioplan, high definition light microscope (Oberkochen, Germany) mounted with a Leica DC 200 digital camera (Heerbrugg, Switzerland).

116 RESULTS

Only hair follicles in the anagen (growth) phase were studied, as hair follicles in catagen (regression) or telogen (resting) phases of the hair cycle were not seen in the sections analysed.

P2Xs, P2 X7 , P2Yi and P 2 Y2 receptors were expressed in human anagen hair follicles

P2 X5 receptor immunoreactivity was observed in the hair kératinocytes of all human skin samples. P2Xg receptor staining was restricted to the medulla and both the inner and outer root sheath (Fig 3.1a, 3.1b), which was continuous with the surface epidermis. P2X? receptor immunostaining was present in the stratum corneum of the overlying epidermis, but there was no staining in the anagen hair follicle (Fig 3.1c, 3.1d). P2Yi receptors were found in the outer root sheath and bulb of anagen hair follicles but not in the inner root sheath (Fig 3.2a, 3.2b). P 2 Y2 receptors were found in cells in the cortex, with possibly a few positive cells at the edge of the medulla, but no staining in the central medulla (Fig 3.2c, 3.2d). Outer root sheath cells positive for P2Yi receptors, also expressed markers for cellular proliferation. Cortex and medulla cells positive for P2 Y2 receptors, did not express markers for proliferation. Double labelling of P2Xs receptors with involucrin showed co-localisation in the inner root sheath and in cells at the edge of the medulla. Double-labelling of P2Yi receptors with Ki67 showed that P2Yi receptors were found in proliferating cells in the outer root sheath and bulb of anagen hair follicles (Fig

3.3a, 3.3b). Double labelling of P2Y% receptors with PCNA showed that P 2 Y2 receptors were expressed in the cortex and possibly at the edge of the medulla (Fig 3.3c) and

PCNA was expressed in the outer root sheath and bulb (Fig 3.3c, 3.3d). P2Y2 receptors were only expressed in living cells and not in the keratinised hair shaft, nor in the central medulla (Fig 3.3d). Involucrin was expressed both in the cortex and inner root sheath, but not in the outer root sheath (Fig 3.3e, 3.3f). P2Xs receptors were expressed in the medulla and in the inner and outer root sheaths. There was co-localisation of involucrin with P 2Xs receptor staining in the inner root sheath and in a few cell layers at the edge of the medulla (Fig 3.3e, 3.3f). Double labelling of P2X? receptors with active caspase-3 did not show any immunoreaction within the anagen hair follicle.

117 Control immunostaining experiments Both the omission of the primary antibody and preabsorption with corresponding peptides were performed as controls. The immunoreaction was abolished after preabsorption of the P2Xs (Fig 3.1e), P2X? (Fig 3.1f), P2Yi (Fig 3.2e) or P 2Y2 antibody (Fig 3.2f) with the corresponding peptides, confirming the specificity of the immunoreaction.

118 DISCUSSION

This study has shown the first direct evidence for the expression of P2Yi, P 2 Y2 and P2X5 receptors in human anagen hair follicles, using immunohistochemistry.

Double-labelling of P2Y 1 receptors with proliferation markers Ki67 and PCNA showed that P2Y1 receptors were found in proliferating cells in the outer root sheath (ORS) and bulb in anagen hair follicles. The ORS is established during the early stages of anagen by the downward migration of the regenerating epithelium and then maintains itself (in contrast to the 1RS and hair shaft), independent of the bulbar matrix by basal cell growth (Reynolds and Jahoda 1991). The thickness and the cellularity of the ORS varies with the level of the follicle; it is single layered near the bulb, higher up it is composed of multilayered cuboidal cells and from the level of the sebaceous gland upwards, it becomes multilayered and is structurally similar to the epidermis (Stenn and Paus 2001). Previous work on normal interfollicular epidermis has shown that P2Yi receptors show a strong immuno-positive signal in the basal layer and double-labelling of P2Yi receptors with keratinocyte proliferation markers Ki-67 and PCNA in the interfollicular epidermis confirmed the presence of P2Yi receptors in proliferating cells. The P2Yi receptor selective agonist, 2MeSADP, caused an increase in human keratinocyte number in vitro (see Chapter 2). It is therefore consistent that P2Yi receptors would have a proliferative role in the anagen hair follicle outer root sheath.

In anagen hair follicles, P 2Y2 receptors were confined to a spatially distinct group of cells in the cortex and at the edge of the medulla, where there was no double labelling with proliferation markers. Commitment to the formation of the three hair shaft cell lineages (medulla, cortex, cuticle) takes place in a sub-compartment of the hair matrix known as the precortex (Niemann and Watt 2002). P 2 Y2 receptors seemed to be expressed on early differentiated cells in the hair shaft, because they were found on living cells of the cortex, but not in the differentiated hair cuticle. Previous work on normal interfollicular epidermis has shown that P 2 Y2 receptors are expressed in all the cells of the basal layer and also in a few cells within the stratum spinosum (see Chapter 2). Stem cells in undamaged epidermis divide infrequently and it is the non-stem daughters of stem cells, ie transit-amplifying cells, which are actively dividing

(Niemann and Watt 2002). Double-labelling of P 2 Y2 receptors with keratinocyte proliferation markers Ki-67 and PCNA in the interfollicular epidermis confirmed the presence of P 2 Y2 receptors in proliferating cells (see Chapter 2). The P 2Y2 receptor agonist, UTP also caused significant proliferation in kératinocytes (Dixon et al. 1999;

119 see Chapter 2) and HaCaT kératinocytes (Lee et al. 2001). P 2Y2 receptors stained all the different cell populations within the basal layer of the epidermis (ie the stem cells,

transit-amplifying cells and post-mitotic cells). P 2 Y2 receptors may then cause proliferation in transit-amplifying cells in the epidermis. Although it is convenient to think of stem and transit-amplifying cells as discrete populations from fully differentiated kératinocytes, there might be gradients of cell behaviour, ranging from a cell that has maximum self-renewal and zero differentiation capacity to one that has completed terminal differentiation and cannot divide again. Transit-amplifying cells would lie in the middle of each gradient (Niemann and Watt 2002). In anagen hair follicles, P 2 Y2 receptors are expressed on early differentiated hair kératinocytes that contribute to the formation of the hair shaft. Cells positive for P 2 Y2 receptors in hair follicles could still have some proliferative capacity, given the right stimulus. In anagen hair follicles, P2Xs receptors were found on cells in the inner and outer root sheaths, and in the medulla. P2Xs receptors are expressed in the basal layer of the interfollicular epidermis, but are expressed more strongly in the stratum spinosum and variably into the stratum granulosum (see Chapter 2). The expression of P2Xs receptors in the outer root sheath was similar to that seen in the epidermis, with basal cells labelling less strongly than suprabasal cells. It has been proposed that P2Xs receptors are involved in human epidermal keratinocyte differentiation (Groschel- Stewart et al. 1999a; see Chapter 2). There is evidence from other tissues regarding the role of P 2X5 receptors. In fetal rat skeletal muscle, P2Xs receptors are sequentially expressed during development (Ryten et al. 2001) and associated with differentiating cells (Ryten et al. 2002). P2Xs receptors have also been implicated in the regulation of osteoblastic differentiation and proliferation (Hoebertz et al. 2000). Involucrin is a marker of keratinocyte differentiation (Watt 1983), which is found in the epidermis in the upper stratum spinosum and in the stratum granulosum. In the hair follicle, involucrin is expressed in the hair cortex, medulla, inner root sheath, and in the innermost cells of the lower outer root sheath (de Viragh et al. 1994). Involucrin co localised with P 2X5 receptor staining in the inner root sheath and in a few cell layers at the edge of the medulla. This suggests a role for P2Xs receptors in the differentiation of cells of the medulla and inner root sheath. There was no staining with antibodies to P2X? receptors or active caspase-3 in the anagen hair follicle. Further work on the expression of P2X? receptors in different stages of the hair cycle would be of interest. Periods of hair growth (anagen) are followed by a regression phase (catagen), when the lower part of the hair follicle

120 undergoes programmed cell death (Cotsarelis 1997), and a resting phase (telogen), before onset of a new growth phase. In this study, only hair follicles in the anagen phase were studied, as hair follicles in catagen or telogen phases of the hair cycle were not found in the sections analysed. At any one time, 84% of scalp hairs are in anagen, 2% in catagen and 14% in telogen (Dawber 1991). The P2X? receptor is unlike other P2X receptors because it is a bifunctional molecule that in addition to forming a channel permeable to small cations, can be triggered to form a cytolytic pore permeable to large hydrophilic molecules up to 900 Da (Surprenant et al. 1996). The opening of this pore results in the increase in intracellular cytosolic free calcium ions and the induction of cell death (Zheng et al. 1991; Ferrari et al. 1996). P2X? receptors are also found on dendritic cells, macrophages and microglial cells, where extracellular ATP can trigger apoptosis via these receptors and there is increasing evidence that this process is dependent on the caspase signalling cascade (Coutinho-Silva et al. 1999; Ferrari et al. 1999). It would be of interest to see if P2Xv receptors are involved in the process of programmed cell death that occurs during the catagen phase of the hair cycle. In summary, P2 purinergic receptors are likely to be involved in outer root sheath cell proliferation via P2Yi receptors found in the bulb and outer root sheath; inner root sheath and medulla differentiation via activation of P2Xs receptors; and early differentiation of cells of the cortex and medulla of the hair shaft via activation of P2Y% receptors. The expression of P2 purinergic receptors in the anagen hair follicle may have therapeutic implications for the treatment of hair loss. The expression of P2 receptors in different cell lineages of the anagen hair follicle could provide the prospect of engineering hair follicles in those with hair loss or excess growth, by using purinergic receptor agonists and antagonists. Lack of growth reflects follicular dynamics and represents the central mechanisms of most common causes of alopecia. In androgenetic alopecia the hair shaft diameter is reduced and there is a progressive decrease in anagen hair percentage, as well as a significant reduction in proliferating cells (Prieto et al. 2002). The anagen phase is also shorter (Hoffmann 2002). This results in the formation of progressively thinner and shorter hair. In some men, there is also a prolongation of the interval between shedding of the club hair in telogen and the emergence of a replacement hair in the anagen phase, giving rise to an increase in the number of ‘empty’ follicles (Courtois et al. 1995). Functional work on hair follicle cycling, cell proliferation and differentiation using purinergic receptor agonists and antagonists may lead to new approaches in the treatment of hair loss.

121 FIGURE LEGENDS

Figure 3.1: Expression of P2Xs and P2X? receptors in human anagen hair follicles.

(a) Longitudinal section of anagen hair follicle. P 2X5 receptor immunostaining was present in the outer root sheath (ORS) and medulla (M), but not in the dermal papilla (DP). Scale bar 75pm. (b) Transverse section of anagen hair follicle. P2Xg receptor immunostaining was present in both the inner root sheath (1RS) and the outer root sheath (ORS) but not in the cortex (CTX) or in the acellular medulla (M) of the hair shaft at this level. Scale bar 30pm. (c) P2X? receptor immunostaining (arrow) was present in the stratum corneum. There was no immunoreaction in the anagen hair follicle. Scale bar 75pm. (d) P2X? receptor immunostaining- transverse section. There was no staining in the outer root sheath (ORS), or cortex (CTX), or acellular medulla (M) of the hair shaft at this level. Scale bar 30pm. (e, f) Controls: the immunoreaction was abolished after preabsorption of the (e) P2Xs and (f) P2X? receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars 100pm.

122 a

ORS

M 2r^ CTX

123 Figure 3.2: Expression of P2Yi and P2Y2 receptors in human anagen hair follicles.

(a) P2Y1 receptors were found in the outer root sheath (ORS) and bulb of anagen hair follicles in longitudinal section, but not in the inner root sheath (1RS). Scale bar 40pm, (b) In transverse section, P2Yi receptors were only seen in the outer root sheath (ORS). Scale bar 40pm. (c) P2Y% receptors were found in the cortex (CTX). Scale bar 40pm.

(d) Transverse section of anagen hair follicle: P 2 Y2 receptors were seen in the cortex (CTX), but not in the central medulla (M), inner (1RS) or outer root sheaths (ORS), or in the surrounding adventitial layer (A). Scale bar 40pm. (e, f) Controls: the immunoreaction was abolished after preabsorption of the (e) P2Yi and (f) P 2 Y2 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars 100pm.

124 1RS ORS

bulb

(T\

125 Figure 3.3: Double-labelling of P2Yi and PZYz receptors with markers for cellular proliferation and double-labelling of P2Xs receptors with markers for keratinocyte differentiation in anagen hair follicles. (a) Double-labelling of P2Yi receptors (red) with Ki67, a nuclear marker for proliferating cells (green), to show that P2Y% receptors were found in proliferating basal cells in the outer root sheath (ORS) and bulb region of the hair follicle in longitudinal section. Scale bar 50pm. (b) Transverse section: double-labelling of P2Yi receptors (red) with Ki67, a nuclear marker (green), to show that P2Yi receptors were found in proliferating cells in the outer root sheath (ORS) of the hair follicle. Scale bar 50pm. (c) Longitudinal section of anagen hair follicle through the dermal papilla (DP): double labelling of P 2 Y2 receptors (red) with PCNA, a marker for proliferating cells (green), showed that P 2 Y2 receptors were found in the cortex (CTX) and at the edge of the medulla (M), but not in the central medulla. P 2 Y2 receptors were not found in the matrix (Ma), where cells were positive for PCNA. Scale bar 75pm. (d) Longitudinal section of anagen hair follicle: P 2 Y2 receptors were absent from the keratinised cuticle (Cu) of the hair shaft. PCNA (green) was also found in cells of the outer root sheath (ORS). Scale bar 75pm. (e) Double-labelling of P2Xs receptors (red-brown) with involucrin, a marker for differentiating cells (green). Involucrin was expressed both in the inner root sheath (1RS), cortex (CTX), and in the outermost edge of the medulla

(M). P2X5 receptors were expressed in the inner (1RS) and outer root sheaths (ORS) and in the medulla (M) and matrix cells (Ma). There was yellow co-localisation with P2Xg receptors in the inner root sheath (1RS) and in cells at the outermost edge of the medulla (arrow). The cortex only stained positive for involucrin, not P2Xg receptors. Scale bar 50pm. (f) Transverse section: double-labelling of P2Xs receptors (red) with involucrin (green). Involucrin was expressed in the inner root sheath (1RS) and cortex (CTX), and co-localised (yellow) with P2Xs receptor staining in the inner root sheath (1RS). Scale bar 50pm.

126 127 CHAPTER 4 PURINERGIC SIGNALLING IN HUMAN FETAL SKIN

128 ABSTRACT

We investigated the expression of P2Xs, P2X?, P2Y% and P 2 Y2 receptor

subtypes in 8-11 week old human fetal epidermis in relation to markers of proliferation (PCNA and Ki-67), keratinocyte differentiation (cytokeratin KIO and involucrin) and markers of apoptosis (indicative of keratinocyte terminal differentiation - TUNEL and anti-caspase-3). Using immunohistochemistry, each of the four receptors was expressed in spatially distinct zones of the developing epidermis: P2Yi receptors were found in the basal layer, P2Xs receptors were predominantly in the basal and intermediate layers, both P 2Y2 and P2X? receptors were in the periderm. Co-localisation experiments suggested different functional roles for these receptors: P2Yi receptors in fetal keratinocyte proliferation; P2X$ receptors in fetal keratinocyte differentiation; and P2X? receptors in apoptosis of periderm cells. The role of P 2 Y2 receptors in periderm cells is not known.

129 INTRODUCTION

Purinergic signalling plays a key role in development from the very beginnings of life, indeed from the moment of conception. Adenosine triphosphate (ATP) is obligatory for sperm movement (Yeung 1986) and is a trigger for capacitation, the acrosome reaction necessary to fertilise the egg (Foresta et al. 1992). ATP is now recognised as an important messenger molecule for cell-cell communication, with ATP binding specifically to purinergic receptors (Bumstock 1997; Rale vie and Bumstock 1998). There is increasing evidence that purinergic signalling can have long-term, trophic effects in embryonic development, growth and cell proliferation (Abbracchio and Bumstock 1998; Bumstock 2001b, 2002a). Purinergic receptors are classified into two groups: PI receptors are selective for adenosine and P2 receptors are selective for adenosine 5’-triphosphate (ATP) and adenosine 5’-diphosphate (ADP), which act as extracellular signalling molecules (Bumstock 1978). P2 receptors are sub-divided into P2X and P2Y receptors (Bumstock and Kennedy 1985; Abbracchio and Bumstock 1994). P2X receptors are ligand-gated ion channels, and are activated by extracellular ATP to elicit a flow of cations (Na"^, K^, and Ca^"^) across the plasma membrane. Seven subtypes of P2X receptors are recognised

(Khakh et al. 2001); all P2X 1.7 subunits have been cloned from mammalian species and are capable of assembling into homo- or heteromultimeric receptors (Torres et al. 1999). Seven subtypes of P2Y receptors have been described so far (King et al. 2000; Communi et al. 2001). P2Y receptors are G-protein coupled and the principal signal transduction pathway involves phospholipase C, which leads to the formation of inositol

1,4,5-triphosphate (IP 3) and mobilization of intracellular calcium. IP 3 regulates cell growth and DNA replication (Berridge 1987). P2X receptors are largely viewed as mediators of short term, fast intercellular communication, while a role for P2Y receptors in mediating the trophic effects of ATP is widely recognised (Neary et al. 1996; Abbracchio and Bumstock 1998). Recent studies suggest that P2X receptors can also mediate trophic effects, including cell proliferation, differentiation and apoptosis. P 2X5 receptors have been implicated in the regulation of osteoblastic differentiation and proliferation (Hoebertz et al. 2000) and in triggering the differentiation of skeletal muscle satellite cells (Ryten et al. 2002). P2X? receptors have been shown to mediate ATP-induced apoptosis (Coutinho-Silva et al. 1999; Di Virgilio et al. 2001). ATP is likely to be an important local messenger in the epidermis. Both P2Xs and P2X? receptors are expressed on adult rat cutaneous kératinocytes and functional

130 roles in the regulation of proliferation, differentiation and cell death have been proposed

(Groschel-Stewart et al. 1999a). P 2Y2 receptors, found in the basal layer of normal adult epidermis, are claimed to be involved in keratinocyte proliferation (Dixon et al. 1999). P2Yi receptors are thought to be mitogenic in endothelial cells (Bumstock 2002a). Studies on adult human epidermis and primary keratinocyte cultures have suggested that P2Yi and P2Yz receptors are involved in keratinocyte proliferation, that P2Xs receptors are likely to be involved in keratinocyte differentiation and P2X? receptors are likely to be part of the machinery of end stage terminal differentiation of kératinocytes (see Chapter 2). Human fetal skin is unique in stmcture and function and is in a continuum of change until birth. The transition from the fetal to the mature epidermis of the neonate and adult involves formation of new epidermal layers, invagination and remodelling of epidermal appendages, and finally loss of fetal-specific cell types and onset of adult- type differentiation (see Holbrook and Hoff (1984) for a review). The primitive epidermis is established at 7-8 days when ectoderm and endoderm are defined in the inner cell mass of the implanted blastocyst. At this stage the epidermis is a single layered ‘indifferent ectoderm’. A second epidermal layer forms at the end of the fourth week. The outermost of the two layers is the periderm. The periderm is the transient, protective covering of the epidermis that is sloughed into the amniotic fluid as soon as differentiation of the underlying epidermal layers is complete at 20-24 weeks. Beneath the periderm, basal kératinocytes comprise the single layer of the ‘epidermis proper’. At the end of the ninth week, the embryonic-fetal transition, basal cells divide to give rise to daughter cells that move vertically to form the first intermediate cell layer that characterises the fetal epidermis. Two or three more layers of intermediate cells are added to the epidermis in the second trimester (12-24 weeks). These cells lie between periderm and basal layers. Once a granular layer is established in the sixth month, the intermediate cells become known as spinous cells; thus each layer of the epidermis assumes the adult nomenclature as the tissue assumes the adult characteristics. The first stratum comeum develops at the end of the second trimester (24 weeks). At first it consists of only a few cell layers, but it increases in thickness during the third trimester and, at birth is approximately equivalent to that of the adult. This study demonstrates the distribution of P2X and P2Y receptors in human fetal epidermis for the first time and compares them with the distribution in the adult epidermis. The distribution of these receptors was analysed in relation to known markers of proliferation, differentiation and apoptosis. This study proposes that these

131 receptors are part of the normal mechanisms controlling fetal epidermal development in relation to keratinocyte proliferation, differentiation and apoptosis.

132 MATERIALS AND METHODS

Tissues Twenty five samples of normal human fetal skin were examined from 9 fetuses

at 8 -11 weeks estimated gestational age (EGA). Samples were obtained from fully informed, consenting patients undergoing elective terminations of pregnancy. Ethics Committee approval was obtained to harvest human adult and fetal samples. Samples of adult, human skin were examined immunohistochemically for comparison. Tissue was frozen in isopentane pre-cooled in liquid nitrogen. Blocks were sectioned at 10pm on a cryostat (Reichert Jung CM1800), collected on gelatin coated slides and air-dried at room temperature. The slides were stored at -20°C. Antibodies The immunogens used for production of polyclonal P2Xg and P2Xv antibodies were synthetic peptides corresponding to 15 receptor-type-specific amino acids (AA) in the intracellular C-termini of the cloned rat P2X receptors, as described previously (Groschel-Stewart et al. 1999a). P2Xg and P2X? antibodies (provided by Roche Bioscience, Palo Alto, California, USA) were kept frozen at a stock concentration of

1 mg/ml. Polyclonal anti-P2Yi and P 2 Y2 antibodies were obtained from Alomone Labs (Jerusalem, Israel), and corresponded to the third extracellular loop of the P2Yi (AA 242-258) and P2Ya receptor (AA 227-244). Antibodies were kept frozen at a stock concentration of 0.6mg/ml (P2Y%, P 2 Y2). Proliferating cell nuclear antigen (PCNA) is a marker for proliferation in normal adult human kératinocytes (Miyagawa et al. 1989). Cytokeratin KIO and involucrin are markers for keratinocyte differentiation (Eckert et al. 1997). PCNA (monoclonal anti-proliferating cell nuclear antigen, clone PCIO, raised in mouse ascites fluid; Sigma, Poole, UK), cytokeratin KIO (BioGenex, San Ramon, CA, USA) and involucrin (Sigma) antibodies were raised in mouse. Ki-67 antigen is a marker for cell proliferation in normal human kératinocytes (Gerdes et al. 1991; Tucci et al. 1998). Active caspase-3 is part of the apoptotic machinery of the cell and is expressed in terminally differentiating kératinocytes (Weil et al. 1999). Ki-67 (DAKO, Denmark) and active caspase-3 (Abeam, Cambridge, UK) antibodies were both raised in rabbit. Immunohistochemistry For immunostaining of cryostat sections, the avidin-biotin technique was used according to a revised protocol (Llewellyn-Smith et al. 1992, 1993). Air-dried sections

133 were fixed for 2 minutes in 4% formaldehyde in O.IM phosphate buffer, containing 0.2% of a saturated solution of picric acid (pH 7.4). Endogenous peroxidase was blocked for 10 minutes with 50% methanol containing 0.4% hydrogen peroxide. Non

specific binding sites were blocked by a 20 minute pre-incubation in 10% normal horse serum (NHS) in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, UK),

followed by incubation with the primary antibodies diluted to 1:100 or 1:200 in antibody diluent (10% NHS in PBS + 2.5% sodium chloride (NaCl) at 4°C overnight. Subsequently, the sections were incubated with biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA, USA) diluted to 1:500 in 1% NHS in PBS for 30 minutes, followed by ExtrAvidin peroxidase conjugate (Sigma, UK) diluted to 1:1000 in PBS for 30 minutes at room temperature. After a wash step, a nickel- diaminobenzidine (DAB) enhancement technique was used to visualise the reaction product. Sections were washed three times with PBS after each of the above steps except for after pre-incubation with 10% NHS. After the last wash, sections were dehydrated twice in isopropanol and mounted with EUKITT (BDH Laboratory Supplies, Poole, UK). Control experiments were carried out with primary antibodies omitted from the staining procedure or the primary antibodies preabsorbed with the corresponding peptides. For the preabsorption control, 1.5pl of the anti-P2X antibody (at 1 mg/ml) was incubated with 24pl of the respective peptide at 5mg/ml overnight at 4°C. 275pi of NHS was added to give 300pl of a 5pg/ml concentration of the P2X antibodies. For P2Yi and

Double-labelling techniques

Double-labelling of P2X or P2Y receptor antibodies with either PCNA, cytokeratin KIO, or involucrin. Sections were fixed and incubated with P2X or P2Y antibodies overnight as described above. After a wash step, biotinylated donkey anti-rabbit IgG antibody, diluted 1:500 in 1% NHS in PBS, was then applied for 1 hour followed by Streptavidin Texas red (Amersham International pic., UK), diluted 1:200 in PBS-merthiolate for 1 134 hour at room temperature. Sections were pre-incubated for 30 minutes with 10% normal goat serum (NGS) diluted in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, UK). They were then incubated for 2 hours at room temperature with one of the following antibodies: PCNA antibody (Sigma, UK) diluted 1:1000; mouse anti-human cytokeratin KIO (BioGenex, USA) diluted 1:50; mouse monoclonal anti-involucrin (Sigma, UK) diluted 1:50. After a wash step, the directly labelled secondary antibody goat anti-mouse FTTC (Nordic Immunological Laboratories, Tilburg, The Netherlands) was applied at a dilution of 1:200 for 1 hour and then sections were washed and mounted in Citifluor (Citifluor Ltd., Leicester, UK). P2X or P2Y receptor immunostaining appeared red and PCNA, cytokeratin KIO and involucrin stained green.

Double-labelling of P2Xj receptor antibodies with TdT-mediated dUTP nick end labelling (TUNEL) TUNEL identifies cells undergoing apoptosis by labelling nuclear DNA fragments that have been cleaved during apoptosis (Gavrieli et al. 1992). TUNEL labelling was performed using a kit (Boehringer Mannheim, Germany). After overnight incubation with P2X-/ receptor antibody diluted to 1:200 as above, sections were washed in PBS and then incubated with the TUNEL reaction mixture for 1 hour at 37°C. As a

135 negative control, sections were incubated with the TUNEL Label solution only. After further washes in PBS, sections were incubated for 1 hour with biotinylated donkey anti-rabbit antibody at a concentration of 1:500. Sections were washed in PBS and then incubated for 1 hour with Streptavidin Texas red (Amersham International pic., UK) at a concentration of 1:200. Sections were washed in PBS and mounted in Citifluor (Citifluor Ltd., UK). P2X? receptor immunostaining appeared red and TUNEL labelling was green. Photography The results were photographed using a Zeiss Axioplan, high definition light microscope (Oberkochen, Germany) mounted with a Leica DC 200 digital camera (Heerbrugg, Switzerland).

136 RESULTS

P2Xs, P2 X7, P2Yi and P 2 Y2 receptor expression in human fetal epidermis. P2Xs (Fig 4.1a) and P2X? receptor immunoreactivity (Fig 4.1b) was observed in the epidermal kératinocytes of all human fetal skin samples. P2Xg receptor staining was found mainly in the basal layer and to a lesser extent in the intermediate cell layer. (Fig 4.1c). P2Xs receptors were found in the cell membranes and sometimes the cell cytoplasm, but not in the nucleus. The staining of the stratum basale cell membranes appeared to be polarised, being mainly concentrated on the basal aspects of the cells that were associated with the basement membrane (Fig 4.1c) In the epidermis, P2X? receptor immunoreactivity was associated with cells within the periderm only (Fig 4.1d). At high power, P2X? receptor immunostaining also labelled fetal blood cells. Both the omission of the primary antibody and preabsorption with corresponding peptides were performed as controls. The immunoreaction was abolished after preabsorption of the P2Xs (Fig 4.1e) or P2X? receptor antibodies (Fig 4.1f) with the corresponding peptides, confirming the specificity of the immunoreaction. P2Yi (Fig

4.1g) and P2 Y2 receptors (Fig 4.1b) were expressed in human fetal skin. P2Yi receptors were found in the basal layer of the epidermis (Fig 4.1i). P2Yz receptors were found only in the periderm layer of the epidermis (Fig 4.1j) The immunoreaction was abolished after preabsorption of the P2Y% (Fig 4.1k) or P2Y% antibody (Fig 4.11) with the corresponding peptide, confirming the specificity of the findings.

Comparison of receptor distribution in fetal and adult epidermis.

P2X5 receptors were found in the basal and intermediate layers of the fetal epidermis (Fig 4.1c), and similarly in the adult, P2Xg receptors were found in the basal layer, stratum spinosum and weakly in the stratum granulosum (Fig 4.2a). There was little or no P2Xs receptor staining in the periderm (Fig 4.1a) or in the stratum comeum (Fig 4.2a). In contrast, P2X? receptor staining was found in the periderm in the fetus (Fig 4.1d) and in the stratum comeum in the adult (Fig 4.2b). P2Yi receptor staining was found in the basal layer in both the fetal (Fig 4.1i) and in the adult epidermis (Fig

4.2c). P2 Y2 receptor staining was found in different layers in the fetus than in the adult: in the fetus, P2 Y2 receptors were found in the periderm (Fig 4.1j), but in the adult, P 2Y2 receptors were found in the basal layer and in the stratum spinosum (Fig 4.2d).

137 Double-labelling of P2Yi receptors with markers for cellular proliferation. Fetal skin was dual stained for Ki-67 and P2Yi receptors (Fig 4.3a) and PCNA

and P2 Y2 receptors (Fig 4.3b), The proliferation markers identified a proliferating subpopulation of basal kératinocytes. Cells positive for these two markers were also

positive for P2Yi but not for P2 Y2 receptors, which were only found in periderm cells.

Double-labelling of P2Xs receptors with markers for keratinocyte differentiation. Double-labelling of P2Xg receptors with cytokeratin KIO (Fig 4.3c) showed that

P2X5 receptors were expressed in differentiating kératinocytes within the fetal epidermis. Cytokeratin KIO, an early marker of keratinocyte differentiation was found in cells in the intermediate cell layer and not in basal kératinocytes. The basal layer stained only for P2Xs receptors, indicating that no differentiation was taking place in these cells. Involucrin, a late marker of keratinocyte differentiation in adult skin was expressed only in the periderm cells and did not co-localise with P2Xs receptor staining (Fig 4.3d). Double-labelling of P2X? receptors with markers for apoptosis in fetal epidermis show co-localisation. Individual rounded periderm cells had nuclei that stained strongly positive for TUNEL (Fig 4.3e). This corresponded to a transitional stage when the periderm is beginning the formation of a series of complex surface blebs. Double-staining of the

P2X7 receptor and TUNEL showed that only intermittent cells were positive for

TUNEL, although P 2X7 receptors were expressed in most of the cells in the periderm layer. The negative control for TUNEL showed no reaction. Double labelling of P 2X7 receptors with anti-caspase-3 showed co-localisation within the periderm (Fig 4.3f).

138 DISCUSSION

This study has shown the first direct evidence for the expression of P2Xs, P2X?,

P2Yi and P2 Y2 receptors in 8-11 week old human fetal epidermis, using immunohistochemistry. Four periods of human epidermal development have been identified (Dale et al. 1985). The first is the embryonic period (before 9 weeks estimated gestational age (EGA)) and the others are within the fetal period: stratification (9-14 weeks), follicular kératinisation (14-24 weeks), and interfollicular kératinisation (beginning approximately 24 weeks). We have studied epidermal tissue at the end of the embryonic period and during the period of stratification.

P2Y1 receptors showed a strong immuno-positive signal in the basal layer of the fetal epidermis, which is known to be where stem cells are found (Bickenbach and Holbrook 1987). Double-labelling of P2Yi receptors and keratinocyte proliferation markers Ki-67 and PCNA was performed. This confirmed the presence of P2Yi receptors in proliferating cells, indicating that this receptor is involved in keratinocyte proliferation. This distribution of P2Y% receptors is the same as we found in adult epidermis.

P2X5 receptors were expressed in the basal layer of human fetal epidermis, and were also expressed in the intermediate cell layer, where cytokeratin KIO, an early marker of keratinocyte differentiation was found. It is known that kératinocytes that initially occupy the intermediate layer of fetal epidermis from 9 to 20 weeks gestation undergo only an incomplete type of differentiation (Holbrook 1991) that is not characteristic of the mature epidermis. Although not morphologically different, the intermediate cells at this stage have initiated biochemical differentiation, characterised by the synthesis of epidermal differentiation-specific markers, keratins 1 and 10 (Dale et al. 1985) as seen in our study. The expression of these keratins is thought to herald the commitment of the keratinocyte to a programme of epidermal keratinocyte differentiation (Haake and Cooklis 1997). Double-labelling of P2Xg receptors with cytokeratin KIO showed that P2Xg receptors were expressed in these differentiating kératinocytes within the fetal epidermis. The basal layer stained only for P2Xs receptors and not cytokeratin KIO, indicating that no differentiation was taking place in these cells. It seems likely, therefore, that P2Xs receptors are involved in the differentiation of epidermal kératinocytes. There is evidence from other tissues regarding the role of P 2X5 receptors. In fetal rat skeletal muscle, P2Xs receptors are sequentially expressed during development (Ryten et al. 2001). P2Xg receptors have been implicated in the regulation

139 of osteoblastic differentiation and proliferation (Hoebertz et al. 2000) and in triggering the differentiation of skeletal muscle satellite cells (Ryten et al. 2002). Involucrin, a late marker of keratinocyte differentiation in adult skin and a comified cell envelope precursor protein, was expressed only in periderm cells and did not co-localise with P2Xs receptor staining. The lack of dual labelling might simply be due to the incomplete nature of epidermal differentiation at this stage in development, since involucrin is a late marker of differentiation. Involucrin has previously been found to be expressed only on periderm cells in fetal epidermis at the same stage of development (Akiyama et al. 1999) and is thought to be representative of a process of kératinisation within the periderm, separate from the kératinisation of the underlying ‘proper’ epidermal layers that occurs later in development. In adult epidermis, we found that involucrin co-localised with P 2Xs receptors only in the upper layers of the stratum spinosum (see Chapter 2). Cells in the periderm were intensely stained for TUNEL and caspase-3. Double staining of the P2X? receptor and TUNEL showed that only intermittent cells were positive for TUNEL, although P2X? receptors were expressed in most of the cells in the periderm layer. Double labelling of P2X? receptors with anti-caspase-3 showed co localisation within the periderm. The co-localisation of P2Xy receptors with two different apoptosis markers, TUNEL and anti-caspase-3 in the periderm is of particular interest. The P2X? receptor is unlike other P2X receptors because it is a bifunctional molecule, that can be triggered to act as a channel permeable to small cations, or on prolonged stimulation can form a cytolytic pore permeable to large hydrophilic molecules of up to 900 Da (Surprenant et al. 1996). The opening of this pore results in the increase in intracellular cytosolic free calcium ions and the induction of cell death

(Zheng et al. 1991; Ferrari et al. 1996). P 2X7 receptors are also found on dendritic cells, macrophages and microglial cells, where extracellular ATP can trigger apoptosis via these receptors and there is increasing evidence that this process is dependent on the caspase signalling cascade (Coutinho-Silva et al. 1999; Ferrari et al. 1999). Caspase-3 is considered an executioner caspase, which once activated will cleave a number of cellular substrates whose limited proteolysis is definitive of apoptosis (Nicholson and Thomberry 1997). Caspase-3 is expressed in adult terminally differentiating kératinocytes (Weil et al. 1999). During apoptosis, the nucleus condenses and DNA is fragmented by endonucleases, which can be detected by TdT-mediated dUTP nick end labelling (TUNEL). TUNEL-positive kératinocytes have been found in the upper regions of the granular layer of the adult epidermis, before comification

140 (Polakowska et al. 1994; Tamada et al. 1994; Gandarillas et al. 1999). Nuclei of individual periderm cells have been previously reported to stain positively for TUNEL from about 14 weeks estimated gestational age, similar to the timing of appearance of transglutaminase I in periderm cells, which is likely to be related to cross-linking proteins like involucrin resulting in the formation of the cell envelope (Polakowska et al. 1994). The periderm is known to exhibit characteristics consistent with apoptosis before its loss from the epidermis (Polakowska et al. 1994), which occurs at midgestation (20-24 weeks). The periderm undergoes a series of morphological changes that have been described ultrastructurally including: blebbing of the cell surface and pinching off of cytoplasmic fragments, accumulation of cytoplasmic filaments, formation of a sub-membranous cell envelope, and a pyknotic nucleus (Holbrook and Odland 1975). These morphological changes in the periderm are similar to those described for other cell types undergoing apoptosis (Kerr et al. 1972) and the time of their onset correlates with the appearance of markers of apoptosis (Polakowska et al. 1994). Following the bleb stage, periderm cells also exhibit a number of regressive changes involving partial organelle degeneration and accumulation of filaments in the cytoplasm. It is thought that apoptosis is the mechanism by which periderm cells are deleted from the epidermis after serving their function (Polakowska et al. 1994). Our study suggests that P2X? receptors are also likely to be part of this apoptotic process and that the expression of P2X? receptors is important in the regulation of apoptosis intrinsic to correct development.

Interestingly, P2 Y2 receptors were also found only in periderm cells. In adult skin, P2Y2 receptors were found in the basal layer of the epidermis, where proliferation markers Ki-67 and PCNA were also found (see Chapter 2). The P 2 Y2 receptor agonist, UTP, also caused a significant increase in human keratinocyte cell number in vitro (see

Chapter 2). Previous work has localised P 2Y2 receptor mRNA in adult human epidermal basal cells via in situ hybridisation (Dixon et al. 1999), in human retinal pigment epithelial cells (Sullivan et al. 1997), equine epithelial cells (Wilson et al. 1998), and frog skin epithelium (Brodin and Nielsen 2000). UTP has also been shown to cause proliferation in kératinocytes (Dixon et al. 1999) and HaCaT cells (Lee et al.

2001). This implied that P 2Y2 receptors were involved in keratinocyte proliferation. There was no staining with proliferation markers PCNA and Ki67 in the periderm cells.

Therefore, the role for P 2Y2 receptors in the periderm still needs to be determined. In summary, P2 purinergic receptors are likely to be involved in fetal keratinocyte proliferation via P2Y 1 receptors found on basal cells and fetal keratinocyte

141 differentiation via activation of P2Xs receptors. P2X? receptors are likely to be involved in apoptosis of periderm cells, but the role of P 2 Y2 receptors in periderm cells is currently unknown.

142 FIGURE LEGENDS

Figure 4.1: P2Xg P2X? P2Yi and P 2 Y2 receptors were expressed in human fetal epidermis

(a) Cross section of hand of 9 week old fetus showing P 2X5 receptor immunostaining. Scale bar 375pm. (b) Cross section of hand of 9 week old fetus showing P2X? receptor immunostaining, with a predominance on the dorsal surface (arrow). Scale bar 375pm. (c) High power view of (a): P2Xg receptor staining was found mainly in the basal layer (B) of the epidermis and to a lesser extent in the intermediate cell layer (I). There was little staining in the periderm (P). Scale bar 25pm. (d) High power view of (b): P 2X7 receptor immunoreactivity was associated with cells within the periderm (P). Scale bar 25pm. (e, f) Controls: the immunoreaction was abolished after preabsorption of the (e)

P2 X5 and (f) P2X? receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars 25pm. (g) Cross section of fingers of 11 week old fetus showing P2Y% receptor immunostaining. Scale bar 425pm. (h) Cross section of fingers of 11 week old fetus showing P 2 Y2 receptor immunostaining. Scale bar 425pm. (i) High power view of (g): P2Yi receptors were found in the basal layer

(B) of the epidermis. Scale bar 25pm. (j) High power view of (h): P 2 Y2 receptors were found only in the periderm (P). Scale bar 25pm. (k, 1) Controls: the immunoreaction was abolished after preabsorption of the (k) P2Yi and (1) P 2Y2 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars 25 pm.

143 p

- L" ,y ,

: , ■ f t r -

144 Figure 4.2: P2Xs, P2X?, P2Yi and P 2 Y2 receptor expression in adult human epidermis.

(a) P2X5 receptor immunostaining of epidermis of adult human leg skin, showing receptors in the stratum basale (SB), stratum spinosum (SS), and weakly in the stratum granulosum (SG), but no staining in the stratum comeum (SC). Scale bar 60pm. (b)

P2X7 receptor immunostaining was positive in the stratum comeum (SC). Scale bar 60pm. (c) P2Yi receptor immunostaining (white), showing positive staining in the stratum basale (SB). Scale bar 30pm. (d) P 2Y2 receptor immunostaining (white), showing positive staining in the stratum basale (SB) and in cells in the stratum spinosum (SS). Scale bar 30pm.

145 S G - ^ ':a

r %

146 Figure 4.3: Double-labelling of P2Yi and P 2 Y2 receptors with markers for cellular proliferation. Double-labelling of P2Xs receptors with markers for keratinocyte differentiation. Double-labelling of P2X? receptors with markers for apoptosis. (a) Double-labelling of fetal epidermis with P2Yi receptors (red) and Ki67 (green) to show that P2Yi receptors were found in proliferating cells in the basal layer (B). Scale bar 25pm, (b) Double -labelling of P 2 Y2 receptors (red) and PCNA (green). PCNA identified the nucleus of a proliferating subpopulation of only basal kératinocytes (B), but P 2 Y2 receptors were only found in periderm cells (P). Scale bar 25pm. (c) Double labelling of P 2X5 receptors (red) with cytokeratin KIO (green). Cytokeratin KIO was found in cells in the intermediate cell layer (I) and not in basal kératinocytes (B). The stratum basale (B) stained only for P 2X5 receptors, indicating that no differentiation was taking place in these cells. Scale bar 25pm. (d) Double-labelling of P2Xs receptors (red) with involucrin (green). Involucrin was expressed only in the periderm cells (P) and did not co-localise with P2Xs receptor staining in the basal (B) and intermediate (I) layers. Scale bar 25pm. (e) Double-labelling of P2X? receptors (red) with TUNEL (green). Only intermittent, rounded periderm cells (P) had nuclei that stained strongly positive for TUNEL although P2X? receptors were expressed in most of the cells in the periderm layer. Scale bar 25pm. (f) Double-labelling of P2X? receptors with anti-caspase-3 showed co-localisation within the periderm (P). Scale bar 25pm.

147 148 SECTION B PATHOLOGICAL TISSUES

149 CHAPTER 5 PURINERGIC RECEPTOR EXPRESSION IN THE REGENERATING EPIDERMIS IN A RAT MODEL OF NORMAL AND DELAYED WOUND HEALING

150 ABSTRACT

This study investigated changes in the expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates are normalised. Excisional wounds were placed within denervated, pedicled, oblique, groin skin flaps and in the contralateral abdomen to act as a control site. Six rats had NGF-treated wounds and six had untreated wounds. Tissue was harvested at day 4 after wounding. The re-epithelialising wound edges were analysed immunohistochemically for P2Xs, P2Xv, P2Yi and P 2Y2 receptors and immunostaining of kératinocytes was quantified using optical densitometry. In normal rat epidermis: P2Y% and P 2Y2 receptors were found in the basal layer where kératinocytes proliferate; P2Xg receptors were associated with proliferating and differentiating epidermal kératinocytes in basal and suprabasal layers; P2X? receptors were associated with terminally differentiated kératinocytes in the stratum comeum. P2Yi receptor expression was significantly increased (P<0.001) in kératinocytes of the regenerating epidermis of denervated wounds but P 2 Y2 receptor expression was significantly decreased (P<0.001). In innervated wounds, NGF treatment enhanced P2Xs (P<0.001) and P2Yi receptor (P<0.001) expression in kératinocytes. In denervated wounds, NGF treatment reduced the expression of P2Yi receptors (P<0.001) in kératinocytes but enhanced the expression of P 2Y2 receptors (P<0.01) compared to untreated denervated wounds. P2X? receptors were absent in all experimental wound healing preparations. P 2X5, P2X?,

P2Yi and P2 Y2 receptor expression in the regenerating epidermis was altered both during wound healing and also by NGF treatment. Possible roles for purinergic signalling and its relation to NGF in wound healing are discussed.

151 INTRODUCTION

This study was designed to investigate changes in the expression of purinergic receptors in epidermal wound healing. Functional roles have been proposed for purinergic receptors in keratinocyte differentiation, proliferation and apoptosis (Groschel-Stewart et al. 1999a; see Chapter 2). All these processes occur in wound healing. We studied the expression of purinergic receptors in normal and denervated wounds, as well as denervated wounds treated with nerve growth factor (NGF). Purinergic receptors are divided into two groups based on different extracellular signalling molecules. PI receptors are selective for adenosine and P2 receptors are selective for adenosine 5’-triphosphate (ATP) and adenosine 5’-diphosphate (ADP) (Bumstock 1978). P2 receptors are sub-divided into P2X (ligand-gated ion channels) and P2Y (G protein-coupled) receptors (Abbracchio and Bumstock 1994). Seven subtypes of P2X receptors (Khakh et al. 2001) and seven subtypes of P2Y receptors have been described (King et al. 2000; Communi et al. 2001). The P2Xs receptor is expressed on kératinocytes undergoing proliferation and differentiation, while the P2Xv receptor is associated with apoptosis. P 2 Yz receptors are found in the basal layer of normal epidermis, and are involved in keratinocyte proliferation (Dixon et al. 1999). P2Yi receptors are thought to be mitogenic in endothelial cells (Bumstock 2002a). Several studies have proposed that extracellular ATP and ADP may have a role in wound healing (Wang et al. 1990). ATP released by damaged cells (Abbracchio and Bumstock 1998) and platelets (Ingerman et al. 1979; George 1985) may be involved in wound healing, tissue repair and regeneration. ATP, acting on purinergic receptors, is involved in cell proliferation in astrocytes and epithelial cells (Neary et al. 1996) and may be an important physiological regulator of epidermal growth and differentiation, acting via inositol triphosphate and intracellular calcium levels (Pillai and Bickle 1992). A rat model of normal and delayed wound healing was chosen. This model was based on the rat pedicled oblique groin flap to produce a denervated wound (Richards et al. 1999). Wound healing has been shown to be delayed in denervated wounds (Richards et al. 1997). The mechanism for this is unknown, and this study was designed to investigate whether P2Xs, P2X?, P2Yi and P2Yi receptors may have a role. It has been shown recently that exogenously applied nerve growth factor (NGF) has a normalising effect on the rate of healing of denervated wounds in a rat model, reducing the time for wound closure (James et al. 2001) and this study aimed to investigate

152 whether the expression pattern of the above purinergic receptors was also normalised by NGF in denervated wounds.

153 MATERIALS AND METHODS

Study design Maintenance and killing of the animals used in this study followed principles of good laboratory animal care and experimentation in compliance with the UK national law and regulations. All animal experimentation was carried out in accordance of the Home Office Guidelines and the Home Office approved Project Licence, (‘Wound Contraction Mechanisms’ Ref. PPL 70/4604). This project was performed in collaboration with S.E. James, who performed all surgical procedures (Personal Licence Ref. PIL 70/14699).

From previous work (Terenghi et al. 1994), it was established that n =6 was the minimum size per group to avoid inter-animal and inter-sample variation influencing the outcome of the results. This group size ensured that the statistical significance was at the 5% level, with an 80% statistical power. Eighteen male adult Lewis rats weighing 200-300 grams were included in this study. The rats were divided into 3 groups of six animals. In group 1, six rats had a denervated flap, which was compared with skin taken from a control site on the contralateral abdomen in the same dermatome. In group 2, six rats had a denervated flap with a wound placed within the flap and a wound placed in the control site on the contralateral abdomen. In group 3, six rats had a denervated flap with a wound treated with NGF placed within the flap and a control wound placed in the contralateral abdomen, also treated with NGF.

Surgical technique

Anaesthesia Induction of anaesthesia was achieved by placing the animal into an induction box and slowly increasing the inspired concentration of halothane (up to 4%) in oxygen. Once anaesthetised the rat was transferred to the operating table where a facemask was used for the maintenance of anaesthesia, again with halothane at between 0.5% and 2% in oxygen with flow rate between 1 and 3 litres per minute, titrated to maintain stable general anaesthesia.

Operation Plastic surgical techniques of atraumatic tissue handling, loupe magnification for general dissection and maximum magnification with an operating microscope for microvascular surgery were observed at all times. The operative site was prepared using

154 clippers to shave the abdominal wall from the costal margin superiorly to the inguinal ligament inferiorly and to both left and right mid-flanks laterally. The skin was prepared for operation using aqueous chlorhexidine 0.05% as an antiseptic. Then the animal was placed in a supine position on the operating table. Normothermia was maintained by placing the animal on an underdrape warming pad covered with a sterile drape. A windowed sterile drape was placed over the prepared skin to maintain sterility and a 50W halogen light source placed overhead the operating field providing both heat and light. Pre-operative skin markings were made in indelible ink with a skin marker. Firstly, the abdominal midline was marked as a dotted line, followed by markings of the groin flap using a 40mm x 20mm template. This flap was positioned at an angle of 70 degrees to the midline, 5mm superior to the inguinal ligament. This flap design lay directly over the natural distribution of the inguinal fat pad. This fat pad was raised as part of the operative procedure and provided a degree of natural ‘insulation’ against reinnervation of the flap from its bed. Using aseptic sterile technique, full thickness skin incisions were made using a size 15 scalpel blade along the marked perimeter of this flap. The flap was then raised by sharp dissection using scissors. The groin flap comprised skin, panniculus camosus, subcutaneous tissue and an underlying fat pad as well as the superficial epigastric vessels. The deep dissection plane was between the abdominal wall musculature and the flap fat pad. The flap was reflected inferiorly so the superficial epigastric vessels could be visualised at their origin from the femoral vessels. The arcade of superficial epigastric vessels could be identified on the undersurface of this axial pattern flap. A warm saline soaked swab was applied to the exposed abdominal musculature and a second warm saline soaked swab was placed over the under surface of the oblique groin flap. Following this, a Zeiss operating microscope was positioned over the pedicle to the flap and its junction with the femoral vessels to enable dissection of the vessels when required. If the operative duration exceeded 60 minutes, a 2.5ml intraperitoneal injection of saline was made to assist with hydration. Following pedicle manipulation the flap was sutured orthotopically using 24 interrupted 5/0 silk sutures spaced at 0.5cm intervals and ensuring careful orientation and skin approximation without tethering of the flap to its bed. In summary, a 40 x 20 mm oblique groin flap was raised on its neurovascular pedicle, containing the superficial epigastric vessels. Sympathetic denervation was achieved by stripping all loose areolar tissue from the pedicle. Compression of the

155 neurovascular pedicle with a clamp for 5 minutes completed the denervation of the flap. The flap was sutured in place orthotopically using interrupted 5/0 silk sutures. A 10 x 10 mm full thickness excisional wound was sited centrally within the flap, with a corresponding size control wound sited on the contralateral side of the abdomen in the same dermatome.

Recovery At the end of the operative procedure the anaesthetic was discontinued and the rat placed in a warmed recovery box at 28°C for 60 minutes. 0.2ml of buphrenorphine (Temgesic®, Sobering Plough) was administered subcutaneously as an analgesic, then the rat transferred to a warmed dedicated recovery room, individually housed in a cage with food and water ad libitam for an overnight stay. Thereafter the animal was returned to the holding room where all animals were kept in uniform conditions of light and dark, temperature and humidity. All operation times and weight of each animal were recorded. The animals were inspected on a daily basis and evaluated for general health and flap viability.

Nerve growth factor (NGF) - treated flaps The oblique pedicled groin flap was raised and denervated, with an excisional wound made as described. Nerve growth factor at 40 [ig/ml in 0.1% bovine serum albumin and PBS was topically applied as a 50 |Li1 aliquot to both denervated wounds and normally innervated control wounds. The rats were anaesthetised for a further 60 minutes to allow the NGF to be absorbed locally. The rats were inspected daily and evaluated for general health and flap viability. No dressing was applied to the wounds and the flaps were harvested at 4 days post-surgery. Previous work has shown that 4 days after denervation, the wound was still open and the flap remained fully denervated (James et al. 2001).

Preparation of primary antibodies The immunogens used for production of polyclonal P2Xg and P2X? antibodies were synthetic peptides corresponding to 15 receptor-type-specific amino acids (AA) in the intracellular C-termini of the cloned rat P2X receptors, as previously described (Groschel-Stewart et al. 1999a)). P2Xg and P2X? antibodies (provided by Roche Bioscience, Palo Alto, California, USA) were kept frozen at a stock concentration of

Img/ml. Polyclonal anti-P2Yi and P 2 Y2 receptor antibodies were obtained from

156 Alomone Labs (Jerusalem, Israel), and corresponded to the third extracellular loop of

the P2Yi (AA 242-258) and P 2 Y2 receptor (AA 227-244). Antibodies were kept frozen

at a stock concentration of 0.6mg/ml (P2Yi, P 2Y2).

Immunohistochemistry Tissue was frozen in isopentane pre-cooled in liquid nitrogen. Blocks were sectioned at 10pm on a cryostat (Reichert Jung CM1800), collected on gelatin-coated slides and air-dried at room temperature. For immunostaining of cryostat sections, the avidin-biotin technique was used according to a revised protocol (Llewellyn-Smith et al. 1992, 1993). Air-dried sections were fixed for 2 minutes in 4% formaldehyde in O.IM phosphate buffer, containing 0.2% of a saturated solution of picric acid (pH 7.4). Endogenous peroxidase was blocked for 10 minutes with 50% methanol containing 0.4% hydrogen peroxide. Non-specific binding sites were blocked by a 20 minute pre incubation in 10% normal horse serum (NHS) in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, Poole, UK). This was followed by incubation with the primary antibodies diluted to 1:100 or 1:200 in antibody diluent (10% NHS in PBS + 2.5% sodium chloride (NaCl) at 4°C overnight. Subsequently, the sections were incubated with biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA, USA) diluted to 1:500 in 1% NHS in PBS for 30 minutes, followed by ExtrAvidin peroxidase conjugate (Sigma) diluted to 1:1000 in PBS for 30 minutes at room temperature. After a wash step, a nickel-diaminobenzidine (DAB) enhancement technique was used to visualise the reaction product. Sections were washed three times with PBS after each of the above steps except after the pre-incubation with 10% NHS. After the last wash, sections were dehydrated twice in isopropanol and mounted with EUKTTT (BDH Laboratory Supplies, Poole, UK). Control experiments were carried out with the primary antibody omitted from the staining procedure and the primary antibodies preabsorbed with the corresponding peptides. For the preabsorption control, 1.5pl of the anti-P2X antibody (at 1 mg/ml) was incubated with 24pl of the respective peptide at 5mg/ml overnight at 4°C. 275pi of NHS was added to give 300pl of a 5pg/ml concentration of the P2X antibodies. For P2Yi and P 2 Y2 antibodies, Ipg antibody was incubated with Ipg peptide: 1.67pl of antibody (at 0.6mg/ml) was added to 2.5pl of peptide (at 0.4mg/ml) and made up to 333pl with NHS, to give a 1:200 solution of P2Y antibody and incubated at 4°C overnight. The antibody/peptide solutions were then agitated and incubated for a further 1 hour at 4°C. The mixture was passed through a

157 syringe filter (0.2|im) and then centrifuged for 5 minutes at 13,000rpm and the supernatant used for immunohistochemistry. The results were analysed using a Zeiss Axioplan high definition light microscope (Oberkochen, Germany) mounted with a Leica DC 200 digital camera (Heerbrugg, Switzerland). Immunohistochemical analysis provided qualitative data.

Optical densitometry To obtain semi-quantitative data, optical density measurements (Charpin et al. 1996) of immunopositive epidermal kératinocytes were performed for sections

immunostained for P2X$, P2Yi and P2 Y2 receptors. To standardise the immunostaining for each antibody, all sections were stained at the same time. Two images of the regenerating epidermis per section were captured at x40 magnification using a BX60 Olympus Microscope (Olympus, Japan) mounted with a spot digital camera. The images were captured at the wound edge, where keratinocyte proliferation was evident but not affected by inflammatory infiltrate or wound debris. The optical density was then calculated using Image-Pro Plus software (Media Cybernetics, Inc, USA) using the formula: OPTICAL DENSITY = -logKINTENSITY- BLACK) / (INCIDENT - BLACK)] ‘INTENSITY’ was the intensity expressed in pixels at the point of study, ‘BLACK’ was the intensity when no light went through the material, and INCIDENT’ was the maximum intensity of the incident light. This formula allowed a Standard Optical density curve to be plotted so that intensity could be converted to optical density and expressed in arbitrary units. A calibration of intensity was performed for both the camera and the images to correct for background before measuring the optical density of each image under study. The optical density of an average of 20 kératinocytes (only cells with a visible nucleus were included) per section was measured, and the mean optical density of immunostained kératinocytes per section was plotted and statistical analyses were performed using GraphPad Prism 3.0 software. One-way analysis of variance (ANOVA) and Bonferroni’s multiple comparison test were carried out between groups for each staining.

158 RESULTS

P2Xs receptor expression, as measured by optical densitometry, was significantly increased (P<0.01) in kératinocytes of the regenerating epidermis in control wounds compared to kératinocytes in unwounded, control epidermis (Figs 5.1a, 5.1b, 5.2). Expression was particularly increased in migratory kératinocytes at the proliferating wound edge (Fig 5.1b) P2Xs receptor expression was significantly increased (P<0.001) in kératinocytes in NGF-treated control wounds (Figs 5.1c, 5.2). P2Xs receptor expression was also increased in kératinocytes of denervated wounds (P<0.001) (Figs 5.1d, 5.2). However, NGF treatment had no statistically significant effect on the expression of P2Xs receptors in the epidermis of denervated wounds (Fig 5.2). So, NGF increased the expression of P2Xs receptors in the epidermis of normally innervated wounds but NGF did not have an effect on the expression of P2Xs receptors in the epidermis of denervated wounds. P2Xv receptor expression was absent in the regenerating epidermis of control wounds compared to unwounded, control epidermis, where the receptor was expressed in the stratum comeum (Figs 5.3a, 5.3b). P 2 X7 receptor expression was absent in the epidermis of five out of the six rats with denervated wounds (Fig 5.3c). P2X7 receptor expression was also absent in the epidermis of both NGF-treated control wounds and NGF-treated denervated wounds. Optical density measurements were not performed due to the very low expression of this receptor in the majority of the sections. P2Yi receptors were found in the basal layer of the epidermis in unwounded, control skin, but in the regenerating epidermis of control wound edges, P2Y 1 receptors were expressed throughout the basal and the suprabasal layers (Figs 5.4a, 5.4b). There was no statistically significant difference between the levels of receptor expression in kératinocytes of control wounds compared to control skin (Fig 5.5). However there was a significant increase (P<0.001) in P2Yi receptor expression in kératinocytes of the regenerating epidermis of NGF-treated control wounds (Figs 5.4c, 5.5). There was also a significant increase (P<0.001) in receptor expression in epidermal kératinocytes of denervated wounds (Figs 5.4d, 5.5). In contrast, NGF treatment of denervated wounds caused a significant reduction (P<0.001) in the expression of P2Yi receptors in kératinocytes (Fig 5.4e) compared to untreated denervated wounds (Fig 5.5). In control skin (Fig 5.4a) there was weak staining of P2Yi receptors in the dermis, immediately adjacent to the overlying epidermis. In NGF-treated innervated control wounds (Fig 5.4c) as well as NGF-treated denervated wounds (Fig 5.4e), there was heavy positive

159 staining of P2Yi receptors in the wound matrix underneath the epidermis. This staining was absent when the primary antibody was omitted and in preabsorption controls of the P2Yi receptor antibody with the corresponding peptide, which were performed on sections of control skin (Fig 5.4f), NGF-treated innervated control wounds (Fig 5.4g) as well as NGF-treated denervated wounds (Fig 5.4h), suggesting that the dermal staining was specific.

P2 Y2 receptors were also found in the basal layer of normal, un wounded epidermis, and the distribution of P 2Y2 receptors was also altered in the epidermal wound edge of healing skin, where they were found throughout the basal and suprabasal layer (Figs 5.6a, 5.6b). The level of P2 Y2 receptor expression, however, was unchanged in kératinocytes in the regenerating epidermis of control wounds compared to kératinocytes from un wounded, control skin (Figs 5.6a, 5.6b, 5.7). There was no significant difference between P 2Y2 receptor labelling in the epidermal kératinocytes of NGF-treated control wounds (Fig 5.6c) compared to the untreated control wounds (Fig

5.7). There was a significant decrease (P<0.001) in P2 Y2 receptor expression in kératinocytes of denervated wounds (Figs 5.6d, 5.7), but there was a significant increase

(P<0.01) in the expression of P2 Y2 receptors in the epidermis of NGF-treated denervated wounds (Fig 5.6e) compared to untreated denervated wounds (Fig 5.7). These results are summarised in Table 5.1. Both the omission of the primary antibody and preabsorption with corresponding peptides were performed as controls. The immunoreaction was abolished after preabsorption of the P2Xs (Fig 5.1e), P2X? (Fig 5.3d), P2Yi (Fig 5.4f) or P 2Y2 (Fig 5.6f) receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreactions.

160 DISCUSSION

This study showed that during wound healing, the expression patterns of purinergic receptors in the epidermis were altered. Kératinocytes proliferate during wound healing, become activated and change to a migratory phenotype (McKay and

Leigh 1991). P 2 Y2 receptors are involved in keratinocyte proliferation (Dixon et al.

1999). P 2Y2 receptor mRNA has been localised in human epidermal basal cells via in situ hybridisation (Dixon et al. 1999). UTP, a P 2Y2 receptor subtype agonist, has also been shown to cause proliferation of normal kératinocytes (Dixon et al. 1999) and HaCaT cells (Lee et al. 2001). P2Y-type receptors can be stimulated with ATP or UTP in the basal layer of frog skin epithelium (Brodin and Nielsen 2000) and on the basolateral membranes of a large number of transporting epithelia (Chan et al. 1997;

Furukawa et al. 1997; Inoue et al. 1997; Leipziger et al. 1997). P 2 Y2 receptors also cause proliferation in rat glomerular mesangial cells (Harada et al. 2000) and have mitogenic effects on vascular smooth muscle cells (Hou et al. 2000). P2Y% receptors are thought to be mitogenic in endothelial cells (Bumstock 2002a), and are found in the basal layer of human epidermis and can be stimulated to cause an increase in keratinocyte number (see Chapter 2). In this study, the level of expression of both P2Yi and P2 Y2 receptors was unchanged in the epidermis of control wound edges compared to normal skin. However, the distribution of the receptors within the epidermis was altered, with both receptors expressed in suprabasal as well as basal kératinocytes at the epidermal wound edge. This could represent part of the change of phenotype that kératinocytes undergo in order to become migratory during the wound healing process. The expression of P2Xs receptors in kératinocytes was significantly increased (P<0,01) during wound healing. P2Xs receptors are found in proliferating and differentiating kératinocytes in rat epidermis (Groschel-Stewart et al. 1999a), but are thought to be more involved in keratinocyte differentiation (see Chapter 2). P2Xs receptors are also found in other stratified squamous epithelia apart from the skin: e.g. cornea, tongue and vagina (Groschel-Stewart et al. 1999a). They are also found in urogenital tract epithelia (Lee et al. 2000), duodenal villus goblet cells (Groschel- Stewart et al. 1999b), as well as in brain, heart, spinal cord and adrenal medulla (Garcia- Guzman et al. 1996). In fetal rat skeletal muscle, P2Xs receptors are sequentially expressed during development (Ryten et al. 2001). P 2X5 receptors have been implicated in the regulation of osteoblastic differentiation and proliferation (Hoebertz et al. 2000) and in triggering the differentiation of skeletal muscle satellite cells (Ryten et al. 2002).

161 The expression of both P2X$ and P2Yi receptors was significantly increased (P<0.001) in kératinocytes of the regenerating epidermis of denervated wounds compared to control wounds, whereas the expression of P 2 Y2 receptors was significantly decreased (P<0.001). This could be explained by either the different nature of the agonists for these receptors or the role of NGF. ATP and ADP act as agonists at

P2X5 and P2Yi receptors respectively, but UTP is a potent agonist at P 2 Y2 receptors. Thus, the receptors might be involved in different processes within the denervated epidermis. The difference in purinergic receptor expression in denervated wounds could be related to the role of NGF. NGF is involved in an autocrine loop within the epidermis to promote keratinocyte proliferation and has a trophic role in cutaneous innervation (Pincelli 2000). The main cellular source of NGF in the skin is basal kératinocytes (Tron et al. 1990). It is possible that in a denervated wound there might be an extra requirement for NGF, because of the need to supply trophic support to nerve fibres growing back into the denervated area. This need could place an extra burden on kératinocytes to synthesise NGF and so one could speculate that in a denervated wound,

P2Xs and P2Yi receptor expression might be increased and P 2 Y2 receptor expression might be decreased when kératinocytes became activated to synthesise NGF. NGF accelerates the rate of normal wound healing (Li et al. 1980;Matsuda et al. 1998). In epidermal kératinocytes from NGF-treated control wounds there was a statistically significant increase (P<0.001) in P2Xs receptor expression compared to untreated control wounds. It seemed that an intact nerve supply was necessary to up- regulate the expression of P2Xs receptors with NGF because there was no statistically significant change in the level of P2Xs receptor expression in kératinocytes of the regenerating epidermis of NGF-treated denervated wounds compared to untreated denervated wounds. NGF has a normalising effect on the rate of healing of denervated wounds (James et al. 2001) which is usually delayed (Richards et al. 1997). The expression of both P2Yi and P 2 Y2 receptors was ‘normalised’ in kératinocytes of the regenerating epidermis of NGF-treated denervated wounds. P2Yi receptor expression in kératinocytes was significantly decreased (P<0.001) in the epidermis of NGF-treated denervated wounds compared to untreated denervated wounds, reducing the level of epidermal expression towards that seen in control wounds. However, in both NGF- treated innervated control wounds and NGF-treated denervated wounds there was heavy positive staining of P2Yi receptors in the wound matrix underneath the epidermis. In control skin there was weak staining of P2Yi receptors in the dermis, immediately

162 adjacent to the overlying epidermis. This staining was absent after preabsorption of the

P2Y1 antibody with the corresponding peptide, suggesting that the dermal staining was specific in both control skin and NGF-treated innervated and denervated wounds. P2Yi receptors are thought to be mitogenic in endothelial cells (Bumstock 2002a), and are expressed on platelets (King et al. 2000), inunune cells (Berchtold et al. 1999; Bumstock 2001a), and fibroblasts (Zheng et al. 1998). These cell types would certainly be present in the underlying wound matrix. It appears that NGF-treatment up-regulated the expression of P2Yi receptors in the wound matrix, and perhaps this might enhance

wound healing. With respect to P 2 Y2 receptors, there was no significant difference between the level of expression of P 2 Y2 receptors in kératinocytes of the regenerating epidermis of NGF-treated denervated wounds and control wounds. P 2 Y2 receptors were significantly increased in epidermal kératinocytes (P<0.01) in NGF-treated denervated wounds compared to untreated denervated wounds. This might also represent a

‘normalisation’ of P 2 Y2 receptor expression in NGF-treated denervated wounds towards the pattem of expression seen in untreated control wounds. During the proliferative phase of epidermal wound healing, keratinocyte proliferation is increased and apoptosis is reduced (Miadelets 1995; Nagata et al. 1999), so that the regenerating epidermis is thickened. P2Xv receptors are strongly linked to apoptosis (Surprenant et al. 1996; Collo et al. 1997). The P2X? receptor can be triggered to form a cytolytic pore permeable to large hydrophilic molecules up to 900 Da (Surprenant et al. 1996). The opening of this pore results in the increase in intracellular cytosolic free calcium ions and the induction of cell death (Zheng et al. 1991; Ferrari et al. 1996). It has been proposed that P2X? receptors are involved in the apoptotic process of terminal differentiation of kératinocytes (Groschel-Stewart et al. 1999a; see Chapter 2). This study showed that the expression of P2X? receptors in kératinocytes at the normal wound edge was reduced compared to normal epidermis and this suggested that the reduction in P2X? receptors was linked to the reduction in apoptosis in the healing epidermis. P2X? receptors were absent in both untreated and NGF-treated experimental wound healing preparations. Autocrine NGF is known to prevent keratinocyte apoptosis (Pincelli 2000). The fact that P2X? receptor expression was absent in both NGF-treated control and denervated wounds would fit with the anti-apoptotic role of NGF, but since P2X? receptors were absent in untreated wounds the receptors would not have been further down-regulated by NGF. NGF is part of an autocrine system regulating epidermal homeostasis (Pincelli 2000). It could be possible that NGF participates in the re-epithelialisation of the wound

163 by up-regulating the expression of P 2X5 and P2Y1 receptors and down-regulating the expression of P2X? receptors in the epidermis. This suggests that these receptors might be involved in the balance between the number of kératinocytes being produced in the basal layer and the number of cells being shed at the cell surface.

In conclusion, P2Xs, P2X?, P2Yi and P 2 Y2 receptor expression in the epidermis was altered during wound healing. P2Yi receptor expression was significantly increased

in kératinocytes of the regenerating epidermis of denervated wounds but P 2 Y: receptor expression was significantly decreased. NGF treatment enhanced P2Xs and P2Y% receptor expression in epidermal kératinocytes of innervated wounds. NGF treatment reduced the expression of P2Yi receptors but enhanced the expression of P2Yz receptors in kératinocytes from denervated wounds, compared to untreated denervated wounds. P2X? receptors were absent in all experimental wound healing preparations. Further work is needed to elucidate the functional role of these receptors in wound healing. Specific agonist and antagonist drugs acting on purinergic receptors may lead to new approaches to wound re-epithelialisation.

164 FIGURE LEGENDS

Figure 5.1: Comparison of P2Xs receptor immunostaining in the epidermis of normal skin, and in the regenerating epidermal wound edge of innervated and denervated excisional wounds and innervated wounds treated with NGF.

(a) Normal control skin from contralateral abdomen. P 2X5 receptors were found in the stratum basale (SB) and in the stratum spinosum (SS) of the epidermis. Scale bar 50pm.

(b) Innervated control wound edge (WE). P 2X5 receptor expression was increased in migratory kératinocytes at the proliferating wound edge. Scale bar 100pm. (c) Innervated control NGF-treated wound edge (WE). NGF increased the expression of

P2X5 receptors in normally innervated wounds. Scale bar 100pm. (d) Denervated wound edge (WE). P2Xs receptor expression was also increased at the edge of denervated wounds. Scale bar 100pm. (e) Preabsorption of normal control skin: the immunoreaction was abolished after preabsorption of the P2X$ receptor antibody with the corresponding peptide, confirming the specificity of the immunoreaction. Scale bar 50pm.

165 SB

' 1 ' , ^ e

166 □ œrtrdskn p<ûom PcûO O l [alccflrdmrd ■ i cortrd \AOJxl + N3F B denervated wxnd ŒIEl denervated vvoLixl + ISCF

FMXOI FMX001

PcûO O l

Figure 5.2: Histogram showing the optical density (arbitrary units) of P2Xs receptor immunostaining in epidermal kératinocytes of different preparations (listed top right) in order to quantify the amount of immunostaining.

167 Figure 5.3: Comparison of P2X? receptor immunostaining in the epidermis of normal skin, and in the regenerating epidermal wound edge of innervated and denervated excisional wounds. (a) Normal control skin from contralateral abdomen. P2X? receptors were found at the junction (large arrow) of the stratum granulosum (SG) and the stratum comeum (SC) and were also found on the outer edge of the stratum comeum (small arrow). Scale bar 50pm. (b) Innervated control wound edge (WE). P2X? receptor expression was absent in control wounds compared to control skin. Scale bar 100pm. (c) Denervated wound edge (WE). P2X? receptor expression was absent. Scale bar 100pm. (d) Preabsorption of normal control skin: the immunoreaction was abolished after preabsorption of the P2X? receptor antibody with the corresponding peptide, confirming the specificity of the immunoreaction. Scale bar 50pm.

168 , f v

' * ■ / # SG \\ / \ SG WE « ^ ac- .. / , X V

•V-S

Ô:

169 Figure 5.4: Comparison of P2Yi receptor immunostaining in the epidermis of normal skin, and in the regenerating epidermal wound edge of innervated and denervated excisional wounds and in both innervated and denervated wounds treated with NGF. (a) Normal control skin from contralateral abdomen. P2Yi receptors were found in the stratum basale (SB) of the epidermis in normal skin and also weakly in the underlying dermis (asterisk). Scale bar 50pm. (b) Innervated control wound edge (WE). P2Yi receptors were also present throughout the suprabasal layers of the epidermis. Scale bar 100pm. (c) Innervated control NGF-treated wound edge (WE). P2Yi receptor expression was increased in the epidermis. Note heavy positive staining in wound matrix (white asterisk). Scale bar 100pm. (d) Denervated wound edge (WE). P2Yi receptor expression was increased in the epidermis. Scale bar 100pm. (e) Denervated NGF-treated wound edge (WE). P2Yi receptor expression was reduced in the stratum basale (SB) of the epidermis compared to untreated denervated wounds. Note heavy positive staining in wound matrix (white asterisk). Scale bar 100pm. (f-h) Preabsorptions controls: (f) normal control skin; (g) innervated control NGF treated wound edge; (h) denervated NGF treated wound edge: the immunoreaction was abolished after preabsorption of the P2Yi receptor antibody with the corresponding peptide, confirming the specificity of the immunoreaction. Scale bars 50pm.

171 P<0.01 0.15i P<0.001 □ control skin l l O control vvcund H control wound + N 3F B denervated wound aio- QED denervated wound + NGF

Figure 5.5: Histogram showing the optical density (arbitrary units) of P2Yi receptor immunostaining in epidermal kératinocytes of the different preparations (listed top right) in order to quantify the amount of immunostaining.

172 Figure 5.6: Comparison of P2Y2 receptor immunostaining in the epidermis of normal skin, and in the regenerating epidermal wound edge of innervated and denervated excisional wounds and in both innervated and denervated wounds treated with NGF.

(a) Normal control skin from contralateral abdomen. P 2Y2 receptors were found in the stratum basale (SB) of normal epidermis. Scale bar 50pm. (b) Innervated control wound edge (WE). P 2 Y2 receptors were also found throughout the suprabasal layers of the epidermis. Scale bar 100pm. (c) Innervated control NGF-treated wound edge (WE).

P2 Y2 receptor expression in the epidermis was unchanged compared to untreated control wounds. Scale bar 100pm. (d) Denervated wound edge (WE). P 2Y2 receptor expression in the epidermis of denervated wounds was reduced compared to control wounds. Scale bar 100pm. (e) Denervated NGF-treated wound edge (WE). P 2Y2 receptor expression was increased in the epidermis compared to untreated denervated wounds. Scale bar 100pm. (f) Preabsorption of normal control skin: the immunoreaction was abolished after preabsorption of the P 2 Y2 receptor antibody with the corresponding peptide, confirming the specificity of the immunoreaction. Scale bar 50pm.

CZI control skin IS l control wxnd WM control wxnd + NGF B dener\ated wxnd Qmi dener\ated wxnd + NGF ao5- 8

aoQ

175 TABLE LEGENDS

Table 5.1: Summary of purinergic receptor subtype expression in epidermal kératinocytes of normal control skin and in the regenerating epidermis of: control wounds, NGF-treated control wounds, denervated wounds and NGF-treated denervated wounds.

Purinergic Receptor Expression in Epidermal Kératinocytes Receptor Control Control Control Denervated Denervated sub-type skin wound wound + NGF wound wound + NGF P2Xs + ++ ++++ +++ +++

P2Xv + 0 0 0 0 P2Yi + + (altered +++ +++ ++ distribution) P2Y2 + + (altered + + distribution)

Key: Level of receptor expression in control epidermis = +; Increased receptor expression compared to control epidermis = ++, +++, ++++; Reduced receptor expression compared to control epidermis = - ; No staining = 0.

176 CHAPTER 6 PURINERGIC SIGNALLING IN NON-MELANOMA SKIN CANCER

177 ABSTRACT

We investigated the use of purinergic receptors as a new treatment modality for non-melanoma skin cancers. Purinergic receptors, which bind ATP, are expressed on human cutaneous kératinocytes. Previous work in rat epidermis suggested functional roles for purinergic receptors in the regulation of proliferation, differentiation and apoptosis. Immunohistochemical analysis of frozen sections in human basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) for P2Xs, P2X?, P2Yi, P 2 Y2 and P2 Y4 receptors was performed, accompanied by detailed analysis of archive material of tumour subtypes in paraffin sections. Functional studies were performed using a human cutaneous SCC cell line (A431), where purinergic receptor subtype agonists were applied to cells and changes in cell number were quantified via a colorimetric assay. Immunostaining in paraffin sections was essentially the same as that in frozen sections, although more detail of the subcellular composition was visible.

P2Xs and P2 Y2 receptors were heavily expressed in BCCs and SCCs. P2X? receptors were expressed in the necrotic centre of nodular BCCs and in apoptotic cells in superficial multifocal and infiltrative BCCs, and SCCs. P2Yi receptors were only expressed in the stroma surrounding tumours. P 2Y4 receptors were found in BCCs but not in SCCs. The P2X? receptor agonist benzoylbenzoyl-ATP and high concentrations of ATP and adenosine (lOOO-SOOOpM) caused a significant reduction in A431 cell number (P<0.001), while the P 2 Y2 receptor agonist UTP caused a significant amount of proliferation (P<0.001). We have demonstrated that non-melanoma skin cancers express functional purinergic receptors and that P2X? receptor agonists significantly reduce cell numbers in vitro.

178 INTRODUCTION

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) of the skin are the commonest malignancies in white people, accounting for greater than 95% of non melanoma skin cancers (NMSC) (Miller and Weinstock 1994), The incidence of NMSCs has risen significantly over the last 10 years (Holme et al. 2000). An estimated 2.75 million cases of non-melanoma skin cancer are diagnosed per year world-wide (Strom and Yamamura 1997), with approximately one million cases per year diagnosed in the USA (Miller and Weinstock 1994). Multiple treatment modalities are available for NMSC, and surgical management is the most common approach. In patients with multiple lesions or in cases of tumours on difficult locations (e.g. eyelids or lips), surgery can cause significant disfigurement. Topical drug treatment is indicated for superficial cutaneous neoplasms, which because of their multiplicity and involvement of large surfaces are difficult to treat with other methods. The purpose of this study was to investigate whether purinergic receptors could represent a suitable target for drug treatment of NMSC. There is increasing evidence that purinergic signalling can have long-term, trophic effects in embryonic development, cell growth, differentiation and proliferation (Abbracchio and Bumstock 1998; Bumstock 2001b, 2002a). Purinergic receptors are classified into two groups: PI receptors are selective for adenosine and P2 receptors are selective for adenosine 5’-triphosphate (ATP) and adenosine 5’-diphosphate (ADP), which act as extracellular signalling molecules (Bumstock 1978). P2 receptors are divided into two main families: P2X and P2Y, based on molecular stmcture, transduction mechanisms and pharmacological properties (Abbracchio and Bumstock 1994). P2Y receptors are G-protein-coupled and the principal signal transduction pathway involves phospholipase C, which leads to the formation of inositol 1,4,5- triphosphate (IP3 ) and mobilization of intracellular calcium. IP 3 regulates cell growth and DNA replication (Berridge 1987). Seven subtypes of P2Y receptors have been described so far (King et al. 2000; Communi et al. 2001). In contrast, P2X receptors are ligand-gated ion channels, and are activated by extracellular ATP to elicit a flow of cations (Na"^, K^, and Ca^"^) across the plasma membrane. Seven subtypes of P2X receptors are recognised (Khakh et al. 2001); all P 2X 1.7 subunits have been cloned from mammalian species and are capable of assembling into homo- or heteromultimeric receptors (Torres et al. 1999).

179 There is growing evidence that ATP may act as an important local messenger in the skin, particularly the epidermis. Previous work has shown that adenosine and ATP have an anti-proliferative effect on cultured human kératinocytes (Cook et al. 1995) and that adenosine Azs receptor mRNA is present on human kératinocytes (Brown et al. 2000). Brown et al. suggested that the mechanism for adenosine mediated growth inhibition was not via an A 2B receptor but via a mechanism involving adenosine transport into the cell (Brown et al. 2000). P2 purinergic receptors are expressed in rat epidermis, on cutaneous kératinocytes, and functional roles in the regulation of proliferation, differentiation and cell death have been proposed (Groschel-Stewart et al. 1999a). In particular, P2Xs receptors are expressed on cells undergoing proliferation and differentiation, while P2X? receptors are associated with keratinised dead cells.

P2Y2 receptors, found in the basal layer of normal epidermis, are claimed to be involved in keratinocyte proliferation (Dixon et al. 1999). P2Yi receptors are thought to be mitogenic in endothelial cells and P 2 Y2 and P2 Y4 receptors stimulate vascular smooth muscle cells proliferation (Bumstock 2002a). There is evidence that there are functional purinergic receptors in human malignant cell lines, including: MCF-7 breast cancer cells (Wagstaff et al. 2000), endometrial cancer cell lines (Katzur et al. 1999), prostate carcinoma cells (Janssens and Boeynaems 2001), colorectal carcinoma cells (Hopfner et al. 1998), ovarian cancer cells (Schultze-Mogasu et al. 2000), and HL-60 leukaemia cells (Conigrave et al. 2000). This study demonstrates the spatially distinct distribution patterns of different P2X and P2Y receptors in human basal cell and squamous cell carcinomas as well as evidence for the actions of purinergic agonists on cell proliferation and death in a squamous cell carcinoma cell line. This suggests a pathophysiological role for these receptors and raises the possibility that purinergic agonists are putative therapeutic agents.

180 MATERIALS AND METHODS

Tissues Frozen sections of ten basal cell carcinomas (5 male, 5 female, mean age 71) and four squamous cell carcinomas (2 male, 2 female, mean age 73) were examined in this study. Ethics Committee Approval was obtained to harvest human non-melanoma skin cancer samples from consenting patients. The difficulty of obtaining fresh, human tissue for frozen sections led to the development of a special method to allow staining of P2Xs

and P2X7 receptors in paraffin sections. Paraffin blocks of a basal cell and squamous cell carcinomas belonging to a collection at Mount Vernon Hospital were examined. Tissue was frozen in isopentane pre-cooled in liquid nitrogen. Blocks were sectioned at 10pm on a cryostat (Reichert Jung CM1800), collected on gelatin coated slides and air-dried at room temperature. The slides were stored at -20°C. Preparation of antibodies The immunogens used for production of polyclonal P2X receptor antibodies were synthetic peptides corresponding to 15 receptor-type-specific amino acids (AA) in the intracellular C-termini of the cloned rat P2X receptors. The peptides were covalently linked to keyhole limpet haemocyanin. The peptide sequences are as follows: P 2X5: AA 437-451, RENAIVNVKQSQILH; P2X?: AA 555-569, TWRFVSQDMADFAEL. The polyclonal antibodies were raised by multiple monthly injections of New Zealand rabbits with the peptides (performed by Research Genetics, Huntsville, Ala., USA). The specificity of the antisera had been previously verified by immunoblotting with membrane preparations from cloned P 2X i_7 receptors expressing CHO K1 cells. The antibodies recognized only one protein of the expected size in the heterologous expression systems, and were shown to be receptor-subtype specific (Oglesby et al.

1999). P 2 X5 and P2X7 receptor antibodies were provided by Roche Bioscience (Palo Alto, California, USA) and were kept frozen at a stock concentration of 1 mg/ml.

Polyclonal anti-P2Yi, P 2Y2 and P2Y4 receptor antibodies were obtained from Alomone Labs (Jerusalem, Israel), and corresponded to the third extracellular loop of the P2Yi (AA 242-258), P2Yz (AA 227-244) and P 2Y4 receptor (AA 337-350).

Antibodies were kept frozen at a stock concentration of 0.6mg/ml (P2Yi, P 2 Y2) and

0.3mg/ml (P 2 Y4).

181 Immunohistochemical methods for frozen sections For immunostaining of cryostat sections, the avidin-biotin technique was used according to a revised protocol (Llewellyn-Smith et al. 1992, 1993). Air-dried sections were fixed for 2 minutes in 4% formaldehyde in O.IM phosphate buffer, containing 0.2% of a saturated solution of picric acid (pH 7.4). Endogenous peroxidase was blocked for 10 minutes with 50% methanol containing 0.4% hydrogen peroxide. Non

specific binding sites were blocked by a 20 minute pre-incubation in 10% normal horse serum (NHS) in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, Poole, UK), followed by incubation with the primary antibodies diluted to 1:100 or 1:200 in antibody diluent (10% NHS in PBS + 2.5% sodium chloride (NaCl) at 4°C overnight. Subsequently, the sections were incubated with biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Lab, West Grove, PA, USA) diluted to 1:500 in 1% NHS in PBS for 30 minutes, followed by ExtrAvidin peroxidase conjugate (Sigma) diluted to 1:1000 in PBS for 30 minutes at room temperature. After a wash step, a nickel- diaminobenzidine (DAB) enhancement technique was used to visualise the reaction product. Sections were washed three times with PBS after each of the above steps except after pre-incubation with 10% NHS. After the last wash, sections were dehydrated twice in isopropanol and mounted with EUKITT (BDH Laboratory Supplies, Poole, UK). Control experiments were carried out with primary antibodies omitted from the staining procedure or the primary antibodies preabsorbed with the corresponding peptides. For the preabsorption control, l.Spl of the anti-P2X antibody (at Img/ml) was incubated with 24pl of the respective peptide at 5mg/ml overnight at 4°C. 275pi of NHS was added to give 300pl of a 5pg/ml concentration of the P2X antibodies. For P2Yi and

P2Y4 antibody 3.34pl antibody (at 0.3mg/ml) was added to 5pi peptide (at 0.4mg/ml) and made up to 333pl to give a 1:200 solution of P2Y antibody and incubated at 4°C overnight. The antibody/peptide solutions were then agitated and incubated for a further 1 hour at 4°C. The mixture was passed through a syringe filter (0.2pm) and then centrifuged for 5 minutes at 13,000rpm and the supernatant used for immunohi stochemi stry.

182 Double labelling of P2 X7 receptor antibody with either TUNEL or anti-human caspase 3 TdT-mediated dUTP nick end labelling (TUNEL) was performed using a kit (Boehringer Mannheim, Germany). TUNEL identifies cells undergoing apoptosis by labelling nuclear DNA fragments that have been cleaved during apoptosis (Gavrieli et al. 1992). Active caspase-3 is part of the apoptotic machinery of the cell and is expressed in terminally differentiating kératinocytes (Weil et al. 1999). Sections were fixed for 2 minutes in 4% formaldehyde prepared in O.IM phosphate buffer, containing 0.2% of a saturated solution of picric acid (pH 7.4). After a wash step, sections were pre-incubated for 20 minutes at room temperature in 10% NHS in PBS-merthiolate, followed by incubation with the P2X? receptor antibody diluted to 1:200 in 10% NHS in PBS-merthiolate at room temperature overnight. Then either the TUNEL or the anti-caspase 3 protocol were followed. For double-labelling with TUNEL, sections were washed in PBS and then incubated with the TUNEL reaction mixture for 1 hour at 37°C. After further washes in PBS, sections were incubated for 1 hour with biotinylated donkey anti-rabbit antibody at a concentration of 1:500. Sections were washed in PBS and then incubated for 1 hour with Streptavidin Texas red (Amersham International pic., UK) at a concentration of 1:200. Sections were washed in PBS and mounted in Citifluor (Citifluor Ltd., Leicester, UK). For double-labelling with anti-capase-3, sections were incubated with biotinylated donkey anti-rabbit IgG (Jackson ImmunoResearch Lab) diluted to 1:500 in 1% NHS in PBS for 1 hour, followed by ExtrAvidin peroxidase conjugate (Sigma) diluted to 1:1500 in PBS for 1 hour, tyramide amplification for 8 minutes (Tyramide Amplification Kit, NEN Life Science Products, Boston, MA) and then Streptavidin Texas red (Amersham International pic., UK), diluted 1:200 in PBS-merthiolate for 10 minutes. Sections were washed three times in PBS after each of the above steps. Sections were pre-incubated for 20 minutes in 10% normal goat serum (NGS) and then incubated at room temperature for 2 hours with rabbit anti-human active caspase-3 (Abeam, Cambridge, UK). Sections were then washed and incubated with the directly labelled secondary antibody, Oregon-green-labelled goat-anti-rabbit IgG (Jackson ImmunoResearch Lab), diluted 1:100 for 45 minutes. Sections were then washed and mounted in Citifluor (Citifluor Ltd., Leicester, UK).

183 Double-labelling ofP2Y2 receptor antibody with PCNA. Proliferating cell nuclear antigen (PCNA) is a marker for cell proliferation in normal human kératinocytes (Miyagawa et al. 1989). Sections were fixed and incubated with P2Y2 antibody diluted 1:100 overnight as described above. After a wash step, biotinylated donkey anti-rabbit IgG antibody, diluted 1:500 in 1% NHS in PBS, was then applied for 1 hour followed by Streptavidin Texas red (Amersham International pic., UK), diluted 1:200 in PBS-merthiolate for 1 hour at room temperature. P 2Y2 receptor immunostaining was observed as a red colour. Sections were pre-incubated for 30 minutes with 10% NGS diluted in O.IM phosphate buffer, containing 0.05% merthiolate (Sigma, Poole, UK), and then incubated for 2 hours at room temperature with PCNA antibody (monoclonal anti-proliferating cell nuclear antigen, clone PC 10, raised in mouse ascites fluid; Sigma) diluted 1:1000. After a wash step, the directly labelled secondary antibody goat anti-mouse FTTC (Nordic Immunological Laboratories, Tilburg, The Netherlands) was applied at a dilution of 1:200 for 1 hour and then sections were washed and mounted in Citifluor (Citifluor Ltd., UK). Immunohistochemical method for paraffîn sections The method below was an adaptation of the routine method used for immunohistochemistry in paraffin sections at RAFT and was developed by Elizabeth Clayton, Histology Department, RAFT. Microwave antigen retrieval was used for the visualization of both P2Xs and P 2X7 receptors in paraffin sections. Paraffin blocks were sectioned at 4pm on a Reichert-Jung Microtome, and sections were taken on Snowcoat Extra slides (Surgipath, St. Neots, Cambs.). The slides were then dried in an oven for 2 hours at 60 °C. P2X? receptors were demonstrated using a routine Streptavidin Alkaline Phosphatase method and a Vector Red final substrate system (Vector Laboratories, Peterborough UK). P2Xs receptors were demonstrated via tyramide amplification and a DAB final substrate system. All histological solvents, buffered formal saline, toluene, xylene and alcohol were supplied by Genta Medical (York, England). Sections were dewaxed and rehydrated using xylene and graded concentrations of industrial methylated spirits, then washed in running tap water for 5 minutes. Sections were microwaved (Daewoo 800W domestic microwave) at full power in a plastic rack for 10 minutes in a covered but not closed container filled with 400mls ImM EDTA at pH 8.0 and then immediately washed in cold tap water for 2-3 minutes. Sections were ringed with a wax pen and placed in a humidification chamber for the application of immunoreagents. After incubation with each immunoreagent, all

184 slides were rinsed off with a wash bottle filled with TBS containing Tween 20 (2 drops Tween per 500mls of TBS) (TBS+T), placed in a slide rack and put in a fresh bath of TBS+T on a magnetic stirrer for 15 minutes. This step was crucial in order to reduce background staining.

Method for P2Xs receptor antibody immunostaining in paraffin sections Endogenous peroxidases were blocked using a 3% hydrogen peroxide solution in methanol containing 0.3% sodium azide for 30 minutes. Sections were washed in running tap water for 2-3 minutes and then placed in a bath of 50mM Tris buffered saline (50mM Tris +150mM NaCl pH 7.6). Sections were incubated with one drop of Avidin D blocking solution for 15 minutes (Avidin Biotin Blocking Kit SP200, Vector Laboratories, Peterborough UK). This was then flicked off and rinsed with TBS+T. One drop of the Biotin blocking solution was then applied for 15 minutes (Avidin Biotin Blocking Kit SP200, Vector Laboratories). Sections were washed with TBS+T and lOOpls 1:5 normal swine serum diluted in DAKO ChemMate antibody diluent (DAKO S2022, DAKO, Ely, UK) was then applied to each section for 30 minutes. Excess swine serum was flicked off the slides and the primary P2Xs receptor antibody (diluted 1:600 in DAKO ChemMate diluent) was applied for 15 minutes. The DAKO CSA kit was used for all subsequent layers as per kit instructions (DAKO Catalysed Serum Amplification system K1500 with biotinylated anti-rabbit secondary antibody (DAKO K1498); DAKO, Ely, UK). After primary antibody incubation and washing, sections were incubated with lOOpls biotinylated anti-rabbit antibody (pre-diluted from the kit) for 15 minutes (Solution 4). After washing with

TBS+T, lOOpls of ABC solution (prepared 30 minutes prior to use (Solutions 5, 6 and 7)) was applied for 15 minutes. After washing with TBS+T, lOOpls of tyramide amplification solution was applied for 15 minutes (Solution 8 ). After a wash step with TBS+T, lOOpls of streptavidin-peroxidase reagent was applied for 15 minutes (Solution 9). Following washing with TBS+T, the DAB substrate chromagen was applied for 5 minutes. P2Xs receptors appeared brown and the intensity of the chromagen was checked under the microscope before washing slides in running tap water.

Method forPlXj receptor antibody immunostaining in paraffin sections

The streptavidin alkaline phosphatase method was used to demonstrate P 2X7 receptors in paraffin sections. This method provided staining with a striking contrast for

185 positive and negative reactions, was fluorescent when looking for low level expression and caused no confusion with endogenous pigments. Endogenous alkaline phosphatase was blocked by a 20 minute incubation in a bath of 20% acetic acid solution. Sections were washed and incubated first with Avidin D blocking solution (Vector Laboratories, Peterborough UK), then Biotin blocking solution (Vector Laboratories, Peterborough UK), and then 1:5 normal swine serum as above. lOOpls of P2X? receptor antibody, diluted 1:100 was applied for 1 hour at room temperature or overnight at 4 °C. After a wash step with TBS+T, lOOpls of biotinylated anti-rabbit antibody (DAKO E0353), diluted 1:200 in DAKO ChemMate diluent (DAKO, Ely, UK), was applied for 30 minutes. After washing in TBS+T, lOOpls of streptavidin alkaline phosphatase (Vector SA5100) diluted 1:200 in DAKO ChemMate diluent, was applied for 30 minutes. After washing in TBS+T, Vector Red substrate (Vector Alkaline phosphatase substrate, SK5100) made up in 200mM Tris HCl (pH 8.2) was applied for 10 minutes. P2X? receptor staining appeared bright pink, the intensity of the chromagen was checked under the microscope prior to washing the slides in running tap water for 2-3 minutes.

Counterstaining Nuclei were counterstained blue with Harris’s haematoxylin for 10-30 seconds and all sections were subsequently dehydrated, cleared and mounted.

Controls Negative controls were used: either diluent only or 1:5 normal swine serum were substituted for the P2Xs or P2X? receptor antibody at an equivalent dilution. Numerous test sections showed no reaction with either the diluent alone or with the added normal swine serum, therefore subsequently the ChemMate diluent was used alone as a negative control. Photography The results were analysed using a Zeiss Axioplan high definition light microscope (Oberkochen, Germany) mounted with a Leica DC 200 digital camera (Heerbrugg, Switzerland). A431 cell line This human epidermal squamous cell carcinoma cell line was purchased from the European Collection of Cell Cultures (ECACC No. 85090402). Cells were grown at

37 °C in a humidified 5% CO2 atmosphere. Cultures were grown on plastic in DMEM

186 supplemented with 10% fetal calf serum (PCS), Penicillin, Streptomycin, and L- glutamine. Cells were disaggregated at 70% confluency with trypsin/EDTA and seeded at 1 X 10"^ cells per well in a 96 well plate. This seeding density gave the best growth curve over a 72-hour period. Proliferation Assay Twenty-four hours after seeding into 96 well plates, medium was gently aspirated from culture wells and PI and P2 receptor subtype agonists and antagonists were applied to the A431 cells, diluted in medium. These included: the PI receptor

agonist, adenosine (Sigma); adenosine 5’-triphosphate (ATP; Sigma); 8 -(p-

sulfophenyl)theophylline ( 8 pSPT; Sigma), A 1/A2 adenosine receptor antagonist (Bruns et al. 1980, 1986); dipyridamole (Sigma), adenosine transport blocking agent (Maguire and Satchell 1979); uridine 5’-triphosphate (UTP; Sigma), P 2 Y2 receptor agonist (von Kiigelgen and Wetter 2000); adenosine 5’-0-(3-thiotriphosphate) (ATPyS; Sigma), P2Xs receptor agonist (Haines et al. 1999b; Khakh et al. 2000; Lambrecht 2000); 2’-& 3’-0- (4-benzoyl-benzoyl) adenosine 5’-triphosphate (BzATP; Sigma), P2X? receptor agonist (Ralevic and Bumstock 1998; Khakh et al. 2001); and 2-methylthioadenosine 5’- diphosphate (2MeSADP; Sigma), P2Yi receptor agonist (von Kiigelgen and Wetter 2000). The pH of the drug solutions prepared in culture medium was 7.0. Changes in cell number were quantified via a colorimetric assay using crystal violet (Gillies et al. 1986; Kueng et al. 1989) and read using a spectrophotometric plate reader (Model 550, Microplate Manager® 4.0 Bio-Rad Laboratories, Inc) at 0, 24, 48 and 72 hours after addition of dmgs. For the colorimetric assay, a solution of 0.5g crystal violet, 0.85g NaCl, 5mls 10% formal saline, 50mls absolute ethanol, 45mls distilled H2O was used. Medium was gently aspirated from wells of a 96 well plate and lOOpl of the colorimetric assay mixture was added to each well and incubated at room temperature for 10 minutes. This mixture allowed simultaneous fixation of cells and penetration of the crystal violet dye into the cells. After washing three times in PBS, 33% acetic acid was used to elute colour from cells and optical density was read at 595nm using the spectrophotometric plate reader. The changes in cell number identified by the crystal violet assay were validated at least once for each drug set by doing actual cell counts with a haemocytometer. To confirm that the optical density of the wells correlated with cell numbers, a control assay was performed for each experiment, where known numbers of cells were seeded in ascending seeding densities and the plate read as soon as cells had attached. Cell number versus optical density was plotted. The R^ value of the trend line was always >0.98.

187 Statistical analysis Each proliferation assay experiment was repeated an average of eight times, each with triplicate samples. Data analysis was performed using Microsoft Excel 97 and GraphPad Prism 3.0 software. One-way analysis of variance (ANOVA) and Bonferroni’s multiple comparison test were carried out between groups.

188 RESULTS

P2Xs, P2Xv, P2Yi, P2Y2 and P 2 Y4 receptors were detected in frozen sections of

basal cell carcinomas.

P2X5 receptors were heavily expressed in tumour cells of basal cell carcinoma,

but slightly less heavily than in the epidermis (Fig 6.1a). At high power, P2Xj receptors were expressed within the cell cytoplasm of tumour cells and the level of staining intensity in tumour cells was comparable to that seen in basal cells within the epidermis.

In the central area of the tumour, staining was absent (Fig 6.1a). P 2 X7 receptors were expressed in the necrotic centre of nodular basal cell carcinomas (Fig 6.1b) and at higher magnification was seen to be associated with mast cells, macrophages and swollen fibroblasts in the stroma surrounding the tumour. Double-labelling of P2X, receptors (red) and TUNEL (green) in the tumour centre showed co-localisation on cells undergoing apoptosis, with P2Xy receptors also expressed in cellular fragments (Fig 6.1c). P2Yi receptors were expressed in the stroma surrounding the tumour, but not within the tumour cells (Fig 6.2a, 6.2b). P2Y2 receptors (Fig 6.2c, 6.2d) and P2 Y4 receptors (Fig 6.2e, 6.2f) were expressed within the tumour but not in the surrounding stromal matrix. Both the omission of the primary antibody and preabsorption with corresponding peptides were performed as controls. There was no immunoreaction when the primary antibody was omitted. The immunoreaction was abolished after preabsorption of the P2Xs (Fig 6.2g), P2X? (Fig 6.2h), P2Yi (Fig 6.2i), P2Y2 (Fig 6.2j) and P2Y4 receptor antibodies (Fig 6.2k) with the corresponding peptides, confirming the specificity of the immunoreaction.

P2Xs, P2 X7 , P2Yi and P2Y% receptors were detected in frozen sections of squamous cell carcinomas.

P2X5 receptors were more heavily expressed in the tumour cells than in the cells of the surrounding epidermis and P2Xs receptors were located specifically on the cell membrane of the squamous cell carcinoma cells (Fig 6.3a), rather than within the cell cytoplasm as in BCC tumour cells. P 2X7 receptors were strongly expressed in the stratum comeum of the epidermis but only weakly expressed in the nucleus of occasional tumour cells (Fig 6.3b). P2Yi receptors were strongly expressed within the stromal matrix surrounding the tumour nests within the dermis, but not expressed within the tumour (Fig 6.3c) P2Y2 receptors were strongly expressed within the tumour, but

189 not expressed within the surrounding stromal matrix (Fig 6.3d). P2 Y4 receptors were not expressed in the SCCs examined. Double-labelling with P2X? receptors (red) and anti-human caspase-3 (green), showed co-localisation in the nuclei of cells (yellow)

within the tumour nests (Fig 6.3e). Double-labelling with P 2 Y2 receptors (red) and PCNA (green) (Fig 6.3f) showed that there was much proliferative activity within the tumour nests and that there was overlap between the cells expressing PCNA and P 2Y2 receptors. Both the omission of the primary antibody and preabsorption with corresponding peptides were performed as controls. There was no immunoreaction when the primary antibody was omitted. The immunoreaction was abolished after preabsorption of the P2Xs (Fig 6.3g), P2X? (Fig 6.3h), P2Yi (Fig 6.3i) and P2 Y2 receptor antibodies (Fig 6.3j) with the corresponding peptides, confirming the specificity of the immunoreaction. The distribution of P2X and P2Y receptors in both basal cell and squamous cell carcinomas is summarised in Table 6.1. P2 receptor staining in normal skin is described in detail in Chapter 2.

P2 X5 and P 2 X7 receptors were expressed in paraffin sections of basal cell and squamous cell carcinomas. The immunostaining of paraffin sections showed essentially the same distribution as that in frozen sections, although more detail of the cellular and subcellular composition was visible. The development of this method also allowed the examination of archive material of paraffin sections of different tumour subtypes. P2Xs and P2Xv receptors were examined because they are involved in early keratinocyte differentiation and terminal keratinocyte differentiation/apoptosis respectively. Paraffin sections of basal cell carcinomas were grouped as follows: 11 nodular (including three solid subtypes), nine superficial multifocal and 24 infiltrative (morpheic). Of the 24 infiltrative BCCs, seven represented a subgroup of ‘horrifying BCCs’ (Horlock et al. 1998), which were clinically locally aggressive and deeply invasive. The distribution of

P2X5 and P2X? receptors within subgroups of basal cell carcinomas is summarised in Table 6.2. In nodular BCCs, P2Xs receptor staining (brown) was present both in the tumour (in the cell cytoplasm), but also in cells of the surrounding stromal matrix (Fig 6.4a). These positive stromal cells were likely to be endothelial cells, seen at higher power in association with capillaries. P2X? receptors (pink) were expressed in the necrotic centre of nodular BCCs (Fig 6.4b) the same as in frozen sections. In superficial multifocal

BCCs, P2X5 receptors were expressed in a patchy fashion throughout tumour nests

190 mostly in the tumour cell cytoplasm (Fig 6.4c) P2X? receptors were weakly expressed in the nucleus of occasional tumour cells (Fig 6.4d). In infiltrative BCCs, the P2Xs receptor staining was different in cells at the edge of the tumour nest, from cells in the centre (Fig 6.4e). Cells at the edge had positive staining in the plasma membrane, and this staining appeared to be polarised to the surface of the cells that were in contact with the basement membrane surrounding the tumour nest. In contrast, cells in the centre of the tumour nest had positive staining for P2Xg receptors in the cell cytoplasm, as seen in nodular (Fig 6.4a) and superficial multifocal BCCs (Fig 6.4c) P2X? receptor immunoreactivity in infiltrative BCCs was mainly in shrunken, condensed, possibly apoptotic nuclei near the centre of tumour nests (Fig 6.4f) In ‘horrifying BCCs’, a locally aggressive and deeply invasive subgroup of infiltrative BCCs, P2Xs receptors were seen in the cytoplasm of tumour cells in association with numerous cells undergoing mitosis (Fig 6.4g). In ‘horrifying BCCs’, P2X? receptors were expressed in the nucleus of numerous cells but unlike in infiltrative BCCs the nuclei were not condensed or apoptotic (Fig 6.4h). Squamous cell carcinomas were sub-classified follows: five Bowen’s disease (carcinomas in situ), four differentiated SCCs and five invasive SCCs. Two basisquamous (metaplastic) carcinomas, showing characteristics of both basal cell and squamous cell carcinomas, were also analysed immunohistochemically. The distribution of P2Xs and P2X? receptors within subgroups of squamous cell carcinomas is summarised in Table 6.3. All examples of Bowen’s disease (squamous cell carcinoma in situ) examined were positive for P2 X5 receptors (Table 6.3) The staining was mainly in the cytoplasm and was much more intense than in the surrounding normal epidermis (Fig 6.5a). There were also positive cells staining for P2Xs receptors within the dermis (Fig 6.5a), which were likely to be endothelial cells in association with capillaries. P2X? receptors were expressed in Civatte bodies (apoptotic kératinocytes) within the tumour (Fig 6.5b) All four well differentiated SCCs were positive for both P2Xg and P2X? receptors (Table 6.3) In well differentiated SCCs, P2Xs receptors were heavily expressed throughout the tumour, in the tumour cell cytoplasm (Fig 6.5c). P 2 X5 receptors were heavily expressed at the edges of keratin pearls, but were less intensely expressed in the more differentiated central areas of keratin pearls. In contrast, P2X? receptors were only expressed in the centre of keratin pearls in more differentiated cells and in areas where necrosis had occurred (Fig 6.5d). In invasive SCCs, P2Xs receptors were more heavily expressed in the invasive tumour compared to normal areas of epidermis (Fig 6.5e). In

191 invasive SCCs, P2X? receptor immunoreactivity was seen within shrunken, pyknotic, apoptotic-looking nuclei, often in cells towards the centre of a tumour nest (Fig 6.5^. In basisquamous (metaplastic) carcinomas, P2Xs receptor labelling was found both in the

tumour cell membrane and within the cytoplasm (Fig 6.5g). P2X7 receptors were expressed in condensed nuclei, which were possibly apoptotic (Fig 6.5h). Both basisquamous carcinomas examined were positive for P2Xg and P2X? receptors (Table 6.3).

P2 X5, P2 X7 , P2Yi and P 2 Y2 receptors were expressed in A431 ceils grown in culture.

P2Xs (Fig 6.6a), P2X? (Fig 6.6c), P2Yi (Fig 6.6e) and P2 Y2 receptors (Fig 6.6g) were expressed on A431 cells on the cell membranes. Both the omission of the primary antibody and preabsorption with corresponding peptides were performed as controls. There was no immunoreaction when the primary antibody was omitted. Furthermore, the immunoreaction was abolished after preabsorption of the P2Xg, P2X?, P2Yi, and

P2 Y2 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction.

Actions of purinergic receptor agonists on cell numbers of cultured cutaneous squamous cell carcinoma, A431 cells. Low concentrations of ATP (lOpM) caused an increase in cell number, whereas high concentrations of ATP (500-5OOOpM) caused a significant decrease (P<0.001) in cell number at 48 hours after application (Fig 6.7), with cells in the 5000pM ATP solution appearing rounded up and dead (Fig 6.7e). At 48 hours after application, the effect of ATP was not significantly different from that of adenosine, apart from at 5000pM, when adenosine had a more potent anti-proliferative effect (Fig 6.8a). The effect of adenosine was not blocked by the adenosine (A 1/A2) receptor antagonist, 8 -(p- sulfophenyl)theophylline (Fig 6.8b) and the adenosine transport blocking agent, dipyridamole had no effect on the adenosine response (Fig 6.8c). The effect of ATP was not significantly reduced by 8 -(p-sulfophenyl)theophylline (Fig 6.9a) and dipyridamole had no significant effect on the response to ATP (Fig 6.9b) Low concentrations of ATPyS (l-lOpM) and high concentrations of UTP (lOOOpM) caused an increase in cell number, with UTP causing a significant effect (P<0.001). High concentrations of ATP (500 pM) and ATPyS (100-500pM) caused a significant decrease (P<0.001) in cell

192 number, at 48 hours after drug application to A431 cells (Fig 6.10) The P2X? receptor agonist, BzATP (500pM) (Fig 6.11) caused a significant reduction in cell number (P<0.001) and the P2Yi receptor agonist, 2MeSADP (l-500pM) (Fig 6.12) caused a reduction in cell number, which was not statistically significant.

193 DISCUSSION

Previous work has shown that adenosine and ATP have an anti-proliferative effect on cultured human kératinocytes (Cook et al. 1995) and that adenosine A 2B receptor mRNA is present in human kératinocytes (Brown et al. 2000). To determine whether PI and P2 receptors were functionally active in A431 cells, the effects of the PI receptor agonist, adenosine and the P2 receptor universal agonist, ATP were investigated. The effect of ATP was not significantly different from that of adenosine, except that at a very high dose (5000pM), adenosine had a more potent anti proliferative effect. The anti-proliferative effect of adenosine was not blocked by the

A 1/A2 receptor antagonist, 8 -(p-sulfophenyl)theophylline, and the adenosine transport blocking agent, dipyridamole had no effect on the response to adenosine. The response to ATP was not blocked by 8 -(p-sulfophenyl)theophylline and dipyridamole had no effect on the response to ATP. These results show that P2 receptor subtypes are present on A431 cells and that adenosine also has a functional role, although this is not mediated by Ai or A2 adenosine receptors. This study focused on experiments to examine the expression and functional roles of P2 receptor subtypes in non-melanoma skin cancers. In this study, the first direct evidence for the expression of P2Xs, P2X?, P2Yi and P2Y2 receptors in human basal cell and squamous cell carcinomas was obtained using immunohistochemistry and functional studies in vitro with the human squamous cell carcinoma, A431 cell line. Low concentrations of ATP increased A431 cell number. Previous work with A431 cells has shown that micromolar range concentrations of ATP are mitogenic for A431 cells (Huang et al. 1989) and increase intracellular calcium levels (Gonzalez et al. 1988). ATP has also been shown to activate cell proliferation in human ovarian tumour cells (Popper and Batra 1993) and in MCF-7 breast cancer cells

(Dixon et al. 1997). The P 2 Y2 receptor agonist, UTP, caused a significant increase

(P<0.001) in A431 cell number in vitro. P 2 Y2 receptors were heavily expressed in both

BCCs and SCCs and there was overlap between cells expressing PCNA and P 2 Y2 receptors. This suggests that P 2 Y2 receptors are involved in tumour proliferation.

Previous work has localised P 2 Y2 receptor mRNA in human epidermal basal cells via in situ hybridisation (Dixon et al. 1999). UTP has also been shown to cause proliferation of normal kératinocytes (Dixon et al. 1999) and HaCaT cells (Lee et al. 2001).

Functional P 2Y2 receptors have also been characterised on human ovarian cancer cells (Schultze-Mogasu et al. 2000), colorectal carcinoma cells (Hopfner et al. 1998), prostate

194 carcinoma cells (Janssens and Boeynaems 2001), endometrial cancer cell lines (Katzur et al. 1999), and MCF-7 breast cancer cells (Wagstaff et al. 2000). In contrast, high concentrations of ATP and the P2X? receptor agonist, BzATP caused a significant decrease in A431 cell number (P<0.001). P2X? receptors were heavily expressed in the necrotic centre of nodular basal cell carcinomas and co localised with TUNEL labelling. They were also expressed in apoptotic cells in both infiltrative BCCs and in squamous cell carcinomas and P2X? receptors co-localised with active caspase-3. The P2X? receptor is unlike other P2X receptors because it is a bifunctional molecule, that can be triggered to act as a channel permeable to small cations, or on prolonged stimulation form a cytolytic pore permeable to large hydrophilic molecules up to 900 Da (Surprenant et al. 1996). The opening of this pore results in the increase in intracellular cytosolic free calcium ions. The P2X? receptor is involved in the release of IL-ip (Ferrari et al. 1997a) and the induction of cell death (Zheng et al. 1991; Ferrari et al. 1996). P2Xv receptors have been localised in rat epidermis (Groschel-Stewart et al. 1999a) and are also found on dendritic cells, macrophages and microglial cells, where high concentrations of extracellular ATP can trigger apoptosis via these receptors and there is increasing evidence that this process is dependent on the caspase signalling cascade (Coutinho-Silva et al. 1999; Ferrari et al. 1999). Extracellular ATP induces apoptosis and inhibits growth of colorectal carcinoma cells in culture (Hopfner et al. 1999). ATP inhibits growth in breast cancer cells (Spungin and Friedberg 1993), androgen-independent prostate carcinoma cells (Fang et al. 1992) and Ehrlich tumour cells (Lasso de la Vega et al. 1994). Basal cell carcinomas are slow-growing tumours with paradoxically high proliferative activity. The slow growth has been previously explained by a concurrent high apoptotic activity (Miller 1991a, 1991b). The interpretation of BCC as a neoplasm with high apoptotic activity has been supported by electron microscopy (Miller 1991a, 1991b) and an end-labelling study (Mooney et al. 1995). From our analysis of different subgroups of BCC in paraffin section, it appears that in moderately aggressive tumours, there is a high level of expression of P2X? receptors, but with increasing aggressiveness of tumour, this is reduced. The 24 infiltrative BCCs studied can be divided into two groups: 17 infiltrative BCCs and seven ‘horrifying BCCs’. In 17 infiltrative BCCs, 59% expressed P2X? receptors but only 14% of ‘horrifying BCCs’ expressed P2X? receptors and within this tumour, the receptor was expressed on most of the cells within it, with positive cells not appearing to be apoptotic, in contrast to the other group of infiltrative BCCs analysed. Reduced apoptosis may lead to unstable tissue kinetics, favouring an

195 increase in total cell numbers, which in tumours correspond to the stage of cellular expansion. A reduction in apoptosis may be responsible for the preservation of genetically aberrant cells, favouring neoplastic progression (Staibano et al. 1999). This may partly explain the aggressive nature of these ‘horrifying BCCs’. Analysis of paraffin sections of squamous cell carcinomas showed that P2Xv receptors were expressed in all the differentiated tumours studied, but in only three out of five invasive SCCs. This would suggest, despite the small sample size that more aggressive tumours

also have reduced expression of P 2X7 receptors.

High concentrations of ATPyS, a potent P 2X5 receptor agonist, caused a significant decrease in cell number, at 48 hours after drug application to A431 cells. This may be because cells are withdrawing from the cell cycle and differentiating. There is evidence from other tissues regarding the role of P2Xg receptors. In normal rat epidermis, P2Xs receptors are found predominantly in differentiating kératinocytes (Groschel-Stewart et al. 1999a). In fetal rat skeletal muscle, P2Xg receptors are sequentially expressed during development (Ryten et al. 2001). P2Xs receptors have been implicated in the regulation of osteoblastic differentiation and proliferation (Hoebertz et al. 2000) and in triggering the differentiation of skeletal muscle satellite cells (Ryten et al. 2002). In paraffin sections of squamous cell carcinomas, P2Xs receptors were highly expressed in all subgroups of tumours, with increased expression compared to the surrounding ‘normal’ epidermis. P2Xs receptors were not found in the basal layer in Bowen’s disease (carcinoma in situ), but only in the suprabasal, differentiated cell layers. P2Xs receptor staining in squamous cell carcinomas was on the cell membrane, whereas in basal cell carcinomas, the receptors were expressed in the cell cytoplasm. In basisquamous (metaplastic) carcinomas, P2Xs receptor labelling was found both in the tumour cell membrane and within the cytoplasm, reflecting the dual differentiation of this tumour subtype. The cytoplasmic distribution and level of staining intensity of P 2X5 receptors in BCC tumour cells was comparable to that seen in basal cells within the normal epidermis, which may reflect the basal cell origin of these tumours. In paraffin sections of BCCs, P 2X5 receptors were expressed on the majority of tumours, however with increasing aggressiveness of tumour, the expression was reduced. Considering the two groups of infiltrative BCCs: out of 17 infiltrative BCCs,

82% expressed P 2X5 receptors but only 57% of ‘horrifying BCCs’ expressed P2Xs receptors. In P2X$ receptor positive ‘horrifying BCCs’, the receptor was expressed on most of the cells within it, in association with numerous cells undergoing mitosis, unlike

196 the other group of infiltrative BCCs analysed, where P2Xs receptor expression was more polarised towards the basement membrane and mitoses were few. P2Yi receptors were expressed in the stroma surrounding both basal cell carcinomas and squamous cell carcinomas, but not within the tumour cells. 2MeSADP, the P2Yi receptor subtype agonist, caused a reduction in A431 cell number, although this was not statistically significant. It is known that for basal cell carcinomas to grow, they depend on interaction with their stromal matrix, and this is why it is generally thought that BCCs are difficult to establish in culture. It seems that P2Yi receptors do not have a direct proliferative role in non-melanoma skin cancers. In summary, P2X? receptor immunostaining may be a useful histological tool to assess apoptosis in tumours, with the loss of expression of the marker suggesting increased tumour aggressiveness. P2 purinergic receptor agonists alter A431 cell numbers via several mechanisms: UTP and low concentrations of ATP cause an increase in cell number via P 2Y2 receptors; ATPyS causes a decrease in cell number, probably because cells are lost from the cell cycle by being induced to differentiate via activation of P2Xs receptors; BzATP and high concentrations of ATP cause a significant decrease in cell number via a direct effect on P2X? receptors, which are known to be involved in mediating apoptosis. We have demonstrated that non melanoma skin cancers contain functional purinergic receptors and agonists can significantly reduce cell numbers. This opens the pathway for new treatment modalities in these important skin cancers.

197 FIGURE LEGENDS

Figure 6.1: P2Xs and P2Xv receptor expression in basal cell carcinoma

(a) P2 X5 receptors were heavily expressed in tumour (T), but slightly less heavily than in the epidermis (arrow). In the central area of the tumour, staining was absent. Scale bar 375pm. (b) P 2 X7 receptors were expressed in the necrotic centre of the basal cell carcinoma (arrowhead), as well as in the stratum comeum (arrow). Scale bar 375pm. (c) Double-labelling of P2X^ receptors (red) and TUNEL (green) in the tumour centre showed co-localisation of cells undergoing apoptosis, with P 2X^ receptors also expressed in cellular fragments. Scale 40pm.

198 ##

199 Figure 6.2: P2Yi, P 2 Y2 and P 2 Y4 receptor expression in basal cell carcinoma

(a) P2Y1 receptors were expressed in the basal layer of the epidermis (arrow) overlying the tumour, and in the stroma (S) surrounding the tumour (T), but not within the tumour itself. Scale bar 100pm. (b) P2Yi receptor distribution within the deeper part of the tumour in the dermis, showing immuno-labelling within the stromal matrix (S) surrounding the tumour (T). Scale bar 100pm. (c) P 2 Y2 receptors were expressed in the basal layer of the epidermis (arrow), and within the tumour (T). There was some nuclear staining within the epidermis Scale bar 100pm. (d) P 2 Y2 receptor distribution within the deeper part of the tumour in the dermis, showing immuno-labelling within tumour (T) and not in the surrounding stromal matrix (S). Scale bar 100pm. (e) P 2Y4 receptors were faintly expressed in the basal layer of the epidermis (arrow), and more strongly within tumour (T). There was some nuclear staining within the epidermis. Scale bar

100pm. (f) P 2 Y4 receptor distribution within the deeper part of the tumour, showing immuno-labelling within tumour (T) and not in the surrounding stromal matrix (S). Scale bar 100pm. The immunoreaction was abolished after preabsorption of the (g)

P2X5, (h) P2X7, (i) P2Yi, (j) P2Ya and (k) P2 Y4 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. There was however some melanin in the basal layer of the epidermis, which could be distinguished from immunostaining by its colour. Scale bars (g-k) 250pm

200 y

^ ^'-': . is .. •■■■ SN ^

##%

y \ S • ® a » s r

%". ifl'"'iir g i

201 Figure 6.3: P2Xs, P2X?, P2Yi and PlYi receptor expression in squamous cell carcinoma a) P2X5 receptors were more heavily expressed on tumour cell membranes (arrows) than on the cells of the surrounding epidermis. Scale bar 30pm. (b) P2X? receptors were strongly expressed in the stratum comeum of the epidermis and only weakly expressed within the tumour (T). Scale bar 30pm. (c) P2Yi receptors were strongly expressed within the stromal matrix (S) surrounding the nests of tumour (T) within the dermis, but not expressed within the tumour itself. Scale bar 50pm. (d) P 2 Y2 receptors were strongly expressed within the tumour (T) within the dermis, but not expressed within the surrounding stromal matrix (S). Scale bar 50pm. (e) Double-labelling with P2X? receptors (red) and anti-human caspase 3 (green), showed co-localisation in the nuclei (yellow) of cells within the tumour nests. Scale bar 50pm. (f) Double-labelling with

P2 Y2 receptors (red) and PCNA (green nuclear staining) showed that there was overlap between the cells expressing PCNA and P 2 Y2 receptors. Scale bar 50pm. The immunoreaction was abolished after preabsorption of the (g) P2Xs, (h) P2X?, (i) P2Y% and (j) P2 Y2 receptor antibodies with the corresponding peptides, confirming the specificity of the immunoreaction. Scale bars (g-j) 200pm

202 V.*-

> T r

C#'-:

m r m m

203 Figure 6.4: P2Xs and P2X? receptors were expressed in paraffîn sections of different subtypes of basal cell carcinoma; sections counterstained with haematoxylin to stain nuclei blue. {a, b) Nodular BCC (a) P2Xs receptors (brown) were expressed within the tumour (T) and cells of the stromal matrix also show strong labelling (arrow). The staining was uniform in nature throughout each tumour nest and mainly in the cell cytoplasm. Scale bar SOpm. (b) P2X? receptors (pink) were expressed in the necrotic centre of nodular BCCs (arrowhead) as in the frozen sections. They were also expressed on inflammatory cells (arrow) surrounding the tumour (T). Scale bar 50pm. (c, d) Superficial multifocal BCC (c) P2Xs receptors (brown) were expressed in a patchy fashion throughout tumour nests mostly in the tumour cell cytoplasm. Scale bar 25pm. (d) P2X? receptors (pink) were weakly expressed in the nucleus of occasional tumour cells (arrows). Scale bar 25pm. (e, f) Infiltrative BCC (e) P2Xs receptors (brown) were expressed in both the basal (arrows) and suprabasal layers of the tumour. Scale bar 50pm. (f) P2X? receptor immunoreactivity (pink) was seen to be nuclear or peri-nuclear (arrows), often in cells towards the centre of a tumour nest. The stained nuclei were condensed and shrunken away from surrounding tissue. Scale bar 50pm. ig, h) ‘Horrifying BCC\ a subgroup of the infiltrative type of BCC, which is locally aggressive, and deeply invasive, (g) P2Xg receptors (brown) were seen in the cytoplasm of tumour cells in association with numerous cells undergoing mitosis (arrows). Scale bar 25pm. (h) P2X? receptors (pink) were expressed in the nucleus of numerous cells (arrows) but unlike infiltrative BCCs (Fig 4f) the nuclei were not condensed. Scale bar 25pm.

204 t

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205 Figure 6.5: P2Xs and P2X? receptors were expressed in paraffin sections of different subtypes of squamous cell carcinoma; sections counterstained with haematoxylin to stain nuclei blue. (a, b) Bowen's disease (Squamous cell carcinoma in situ) (a) P2Xg receptors (brown) were expressed within the tumour (T) and also in cells of the stromal matrix. There was almost no staining within the basal layer of the epidermis (arrow). Scale bar 50pm. (b) P2X? receptors (pink) were expressed in Civatte bodies (apoptotic kératinocytes - arrow) within the area of the tumour. Scale bar 50pm. (c,d) Well differentiated SCC (c) P2Xs receptors (brown) were heavily expressed throughout the tumour, especially in areas where keratin pearls (asterisk) were forming. The receptors were also found in the tumour cell cytoplasm, but were less expressed in the more differentiated areas of the keratin pearls (asterisk). Scale bar 50pm. (d) P2X? receptors (pink) were only expressed in the centre of keratin pearls in the more differentiated cells and in areas where necrosis had occurred (arrow). Scale bar 50pm. (e, f) Invasive SCC (e) P2Xg receptors (brown) were more heavily expressed in the invasive tumour (T) compared to the more normal areas of epidermis nearby (double arrows). Scale bar 100pm. (f) P2X? receptor immunoreactivity was seen in the nucleus

(arrow), often in cells towards the centre of a tumour nest. Scale bar 100pm. (g, h) Basisquamous (metaplastic) carcinoma, (g) P2Xs receptors (brown) were seen in all tumour cells. Scale bar 50pm. (h) P2X? receptors (pink) were expressed in nuclei (arrows) which were condensed and possibly apoptotic. Scale bar 50pm.

206 m m m

207 Figure 6 .6 : P2Xs, P2X?, P2Yi and P2Y% receptor expression in squamous cell carcinoma, A431 cells grown in culture

(a) P2X5 (c) P2 X7 (e) P2Yi (g) P 2 Y2 receptor immuno-labelling on the membranes of A431 cells. Scale bar 20pm. (b, d, f, h) Phase contrast micrographs of the same fields.

Scale bar 20pm.

208 i i K £ .

209 Figure 6.7: Change in cell number of squamous cell carcinoma, A431 cells at 48 hours after ATP application. At 48 hours after application, low doses of ATP (lOpM) caused an increase in cell number, whereas high doses of ATP (1000-5000pM) (P<0.001) caused a significant decrease. Results represent the mean of 26 experiments. *P<0.001 compared to control. Error bars represent mean +/-SEM. These effects were shown also by the colony size and cell morphology. A431 cells were stained with crystal violet and phase contrast micrographs were taken, (a) Control - cells grown in A431 medium only, (b) lOpM ATP caused colonies of A431 cells to appear visibly larger than in control wells, (c) lOOpM ATP caused no change in the colony size, (d) lOOOpM ATP and (e) SOOOpM ATP both caused a marked decrease in colony size, with cells rounding up and dying in 5000|iM ATP. Scale bar 150pm.

210 % change in cell number from control, measured by optical densitometry A 0> N) g o o O O o _l______L_ _ l _ _1_ _L_ f

03 S

o 13 o 1 s, I I

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T 11

drug concentration

rrnrîa Adenoair^

y / A v / / / / / /

drug concentration /jiM

Figure 6 .8 : At 48 hours after application to A431 cells: a) The effect of ATP was not significantly different from that of adenosine, apart from at SOOOpM, when adenosine caused a greater decrease in cell number (P<0.001). b) 30pM 8 -(p- sulfophenyl)theophylline (8 pSPT) caused no significant block of the effect of adenosine, c) 30pM Dipyridamole (Dip) caused no significant effect on the adenosine response.

Results represent the mean of up to 9 experiments. Error bars represent mean +/-SEM.

(*P<0.001 compared to control).

2 1 2 a 20 HATP I 130|iM SpSPT ATP ■5 10 - Il ' m -1 0

i ° -20 = -Q S? c = -30-

i g .40- T

{///%///%/ 4^^

drug concentration /^M

^ATP CZlaOuM Dipyridamole + /- AT P II . a # 75y -10 3 O i s 2 0 - »|.3o

re E -40-

drug concentration /pM

Figure 6.9: At 48 hours after application of either a) 30pM 8 -(p- sulfophenyl)theophylline ( 8 pSPT) or b) 30pM Dipyridamole (Dip), no significant effect on the response to ATP was seen on A431 cell number.

Results represent the mean of up to 9 experiments. Error bars represent mean +/-SEM.

(*?<0.001 compared to control).

213 50i

OATPyS i n UTP 2 5 -

- 2 5 i

- 5 0 -

-7 5 - control 1 1 10 10 100 100 500 500 1000

(*ug concentration /\iM

Figure 6.10: Comparison of effect of ATP with UTP and ATPyS at 48 hours after drug application to A431 cells. At 48 hours after application, ATP (1-1 OpM), ATPyS (1-10 pM) and UTP (lOOOpM) (P<0.001) caused an increase in cell number of primary human keratinocyte cultures, whereas ATPyS (100-500pM) (P<0.001) and ATP (500pM) (P<0.001) caused a significant decrease. Results represent the mean of 8 experiments. *P<0.001 compared to control. Error bars represent mean -+-/-SEM.

214 20 -

10-

0 - 8 g

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- 20 - if -3 0 - = o 8 ^ -4 0 - • S i O) 3 -5 0 - C w (Q (0 x: o ü P -6 0 -

-70^

-80 control 10 100 5 00

BzATP concentration /^iM

Figure 6.11: Percentage change in A431 cell number with BzATP, P2X? receptor subtype agonist, at 48 hours after drug application. BzATP (500pM) caused a significant reduction in cell number (*P<0.001). Results

represent the mean of 8 experiments. *P<0.001 compared to control. Error bars represent mean -I-/-SEM.

215 5-

-5 - II

-1 5 i

-20 control 10 100 5 0 0

2MbSADP concentration /^iM

Figure 6.12: Percentage change in A431 cell number with 2MeSADP, P2Yi receptor subtype agonist, at 48 hours after drug application. 2MeSADP (l-500pM) caused a reduction in cell number, which was not significant.

Results represent the mean of 8 experiments. Error bars represent mean +/-SEM.

216 TABLE LEGENDS

Table 6.1: Distribution of P2X and P2Y receptor subtypes in normal skin, BCCs and SCCs. Receptor Normal skin BCCSCC P2Xs Weaker in basal layer, Tumour cell Tumour cell strong staining in stratum cytoplasm, membranes, spinosum and granular layer endothelial cells endothelial cells kératinocytes

P2X t Stratum comeum Necrotic areas within Nucleus of tumour, mast cells, occasional tumour macrophages and cells fibroblasts P2Yi Basal epidermal Stroma Stroma kératinocytes

P2 Y2 Basal epidermal Tumour cell Tumour cell kératinocytes cytoplasm cytoplasm P2Y4 Weak in epidermis Tumour cell No staining cytoplasm

217 Table 6.2: Analysis ofP2Xs and P2Xj receptor distribution in paraffin sections of basal

cell carcinoma subgroups.

BCC subtype P2Xs P2X7 P2Xs +ve/ P2Xs -ve/ P2Xs +ve/ P2Xs -ve/ +ve +ve P2X? +ve P2X? -ve P2X? -ve P2X? +ve

Nodular (11) 7 4 4 4 3 0

Superficial 5 3 3 4 2 0 multifocal (9)

Infiltrative 14 10 9 2 5 1 (17)

Horrifying (7) 4 1 1 3 3 0

218 Table 6.3: Analysis of P2Xs and PlXy receptor distribution in paraffin sections of squamous cell carcinoma subgroups

SCC subtype P2Xs P2X t P2Xs +ve/ P2Xg -ve/ P2Xs +ve/ P2Xs -ve/ +ve +ve P2X? +ve P2X? -ve P2X? -ve P2Xy +ve Bowen’s 5 3 3 0 2 0 Disease (carcinoma in situ ) (5) Differentiated 4 4 4 0 0 0 (4) Invasive (5) 5 3 3 0 2 0 Basisquamous 2 2 2 0 0 0 (metaplastic) (2)

219 CHAPTER 7 P2Xs AND P2X, RECEPTORS IN HUMAN WARTS AND CIN-612 RAFT ORGANOTYPIC CULTURES OF HUMAN PAPILLOMAVIRUS INFECTED KERATINOCYTES

220 ABSTRACT

This study aimed to investigate the pattern of purinergic receptor staining in cutaneous warts. Purinergic receptors, which bind ATP, are expressed on human cutaneous kératinocytes and in squamous cell carcinomas. Studies on normal human epidermis and functional studies on primary keratinocyte cultures have suggested that P2Xs receptors are likely to be involved in keratinocyte differentiation and P2Xv receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of kératinocytes. P2X? receptor agonists can significantly reduce primary keratinocyte cell numbers in culture. Human papillomaviruses are increasingly being recognised as important human carcinogens in the development of non-melanoma skin cancers. In our study, immunohistochemical analysis for P2Xs and P2X? receptors was performed in paraffin sections of normal human skin, warts, raft organotypic cultures of normal human kératinocytes and raft organotypic cultures of CIN-612 cells, a model of kératinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of P2Xs receptors. A similar pattern was seen in the CIN-612 raft organotypic cultures. Both P2Xs and P2X? receptors were found in the nuclei of koilocytes, abnormal kératinocytes characteristic of human papillomavirus infection. P2Xg and P2X? receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death. Raft organotypic cultures of both normal human kératinocytes and HPV infected cells could prove to be a useful tool for further study of the role of these receptors.

221 INTRODUCTION

Non-melanoma skin cancer (NMSC) is the most frequently occurring malignancy worldwide in the Caucasian population (Miller and Weinstock 1994). Ultraviolet radiation is the major environmental factor in the pathogenesis of BCCs and SCCs, which tend to occur in sun-exposed sites (Frost and Green 1994). The ratio of basal cell carcinomas (BCCs) to squamous cell carcinomas (SCCs) is 5:1 in immunocompetent populations, whereas in immunosuppressed patients the ratio is reversed, with the risk of developing a SCC up to 250 times greater and the risk of developing a BCC ten times greater than that in the general population (de Villiers et al. 1997). The difference in incidence of these tumours in immunocompromised patients is thought to be due to the involvement of papillomaviruses. The association between warts and skin cancer was first noted in renal transplant recipients (Walder et al. 1971). Human papillomaviruses (HPVs) are small double-stranded DNA viruses which are widespread in the human population. They are strictly epitheliotropic and infect only cutaneous and mucosal skin sites. Over 120 different HPV types have been identified, of which 80 have been characterised in full (de Villiers et al. 1997). There is increasing evidence that purinergic signalling can have long-term, trophic effects in cell growth, proliferation, differentiation and death (Abbracchio and Bumstock 1998; Bumstock 2002a). Purinergic receptors are classified into two groups: PI receptors are selective for adenosine and P2 receptors are selective for adenosine 5’- triphosphate (ATP) and adenosine 5’-diphosphate (ADP), which act as extracellular signalling molecules (Bumstock 1978). P2 receptors are sub-divided into P2X and P2Y receptors (Bumstock and Kennedy 1985; Abbracchio and Bumstock 1994). P2X receptors are ligand-gated ion channels, and are activated by extracellular ATP to elicit a flow of cations (Na"^, K^, and Ca^"^) across the plasma membrane. Seven subtypes of P2X receptors are recognised (Khakh et al. 2001). In contrast, P2Y receptors are G- protein coupled and seven subtypes of P2Y receptors have been described (King et al. 2000; Communi et al. 2001). P2X receptors are largely viewed as mediators of short term, fast intercellular communication. Recent studies suggest that P2X receptors could also mediate trophic effects. P2Xs receptors have been implicated in the regulation of osteoblastic differentiation and proliferation (Hoebertz et al. 2000), and triggering the differentiation of skeletal muscle satellite cells (Ryten et al. 2002). P2X? receptors have been shown to mediate ATP-induced apoptosis (Coutinho-Silva et al. 1999; Di Virgilio

2 2 2 et al. 2 0 0 1 ) and have been demonstrated in rat epidermis in association with dying kératinocytes (Groschel-Stewart et al. 1999a).

ATP is likely to be an important local messenger in the epidermis. Both P 2X5 and P2X7 receptors are expressed on adult rat cutaneous kératinocytes and functional roles in the regulation of cell turnover have been proposed (Groschel-Stewart et al. 1999a). Studies on adult human epidermis and primary keratinocyte cultures (see Chapter 2) have suggested that P2Xg receptors are likely to be involved in early keratinocyte differentiation and P2X? receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of kératinocytes. This study compares the distribution of P2Xs and P2X? receptors in normal human skin, with that in human warts for the first time. Vegetative reproduction of HPV particles can only take place in highly differentiated kératinocytes. This has made it difficult to study the effects of HPV in vitro. We studied the distribution of P2Xs and P2X? receptors in HPV infected kératinocytes grown on a collagen raft. These receptors may be a useful target for further research into both HPV and the carcinogenesis of cutaneous SCCs using HPV infected kératinocytes grown on collagen rafts as a research tool.

223 MATERIALS AND METHODS

Tissues Paraffin sections of normal skin, human warts, human keratinocyte (NHEK) raft organotypic cultures (raft cultures) and CIN 612 (HPV 31 infected cell line) raft cultures were stained for P 2X5 and P2X? receptors. The paraffin sections of warts, NHEK raft cultures and CIN 612 raft cultures belonged to a collection from Roche Discovery, Welwyn. P2Xs and P2X? receptors were examined because they are involved in early keratinocyte differentiation and terminal keratinocyte differentiation/apoptosis respectively. Raft organotypic cultures of differentiated HPV infected kératinocytes. The method for raft culture of kératinocytes has already been described (Ozbun and Meyers 1996). Briefly, primary human foreskin kératinocytes (NHEKs) were obtained from Clonetics (San Diego, USA), the CIN-612 cell line was established from a CIN I biopsy and contained HPVBlb DNA. Epithelial cells were seeded onto collagen matrices containing J2 3T3 fibroblast feeders. When the epithelial cells had grown to confluence, collagen matrices were lifted onto stainless steel grids and the cells were fed by diffusion from under the matrix. The cells were allowed to stratify and differentiate at the air-liquid interface over a 16-day period. Raft cultures were then harvested, fixed in 4% paraformaldehyde and embedded in paraffin. Antibodies The immunogens used for production of polyclonal P2Xg and P2X? antibodies were synthetic peptides corresponding to 15 receptor-type-specific amino acids (AA) in the intracellular C-termini of the cloned rat P2X receptors, as previously described (Groschel-Stewart et al. 1999a)). P2Xs and P2X? antibodies (provided by Roche Bioscience, Palo Alto, California, USA) were kept frozen at a stock concentration of

1 mg/ml. Immunohistochemical method for paraffîn sections The method below was an adaptation of the routine method used for immunohistochemistry in paraffin sections at RAFT and was developed by Elizabeth

Clayton, Histology Department, RAFT. The method is described in detail in Chapter 6 .

Briefly, Microwave antigen retrieval was used for the visualization of both P 2X5 and

P2X7 receptors in 4pm paraffin sections. P2Xs receptors were demonstrated via tyramide amplification and a diaminobenzidine (DAB) final substrate system, so that

224 receptors were stained brown. P2X? receptors were demonstrated using a routine Streptavidin Alkaline Phosphatase method and a Vector Red final substrate system (Vector Laboratories, Peterborough UK), so that receptors were stained pink. Nuclei were counterstained blue with Harris’s haematoxylin. Control experiments were carried out with the primary antibody omitted from the staining procedure. Photography The results were analysed using a Zeiss Axioplan high definition light microscope (Oberkochen, Germany) mounted with a Leica DC 200 digital camera (Heerbrugg, Switzerland).

225 RESULTS

P2Xs and P2X? receptors in paraffin sections of normal human skin. P2Xs immunoreactivity was present mainly in the viable cell layers of normal human skin (Fig 7.1a), where the staining was confined largely to the cell membranes and the cytoplasm in epidermal kératinocytes, with some nuclear staining. P2X? immunoreactivity was present in the epidermis of all normal skin samples, and was associated with cells and cell fragments in the stratum comeum (Fig 7.1b). There was some staining of the outermost edge of the stratum comeum with the P2Xs receptor antibody, which was only slightly reduced in the no primary antibody control (Fig 7.1c), and therefore non-specific staining. There was no staining in the no primary control for the P2X? receptor antibody (Fig 7,ld).

P2Xg and P2X? receptors in paraffîn sections of human warts. In warts, there was marked hyperkeratosis and parakeratosis within the stratum comeum. P2Xs receptors were present in nucleated kératinocytes in areas of parakeratosis, but not within the hyperkeratotic areas of the stratum comeum (Fig 7.2a). In contrast, P2X? receptors were found in hyperkeratotic areas of the stratum comeum but not in parakeratotic areas of the wart (Fig 7.2b). P2Xs immunoreactivity was present in the majority of wart kératinocytes (Fig 7.2c), but few cells in the basal layer were positive, with most of the positively stained cells in the suprabasal layers. P2X? receptors were found in the nuclei of suprabasal cells (Fig 7.2d). There was a prominent granular layer in the wart with koilocytes. Koilocytes are the characteristic cytological feature of HPV infection. Koilocytes are kératinocytes with pyknotic, deeply blue nuclei surrounded by a halo and clear cytoplasm with a paucity of keratohyaline granules. They usually indicate the presence of human papilloma vims. Both P2Xg and P2X? receptors were found in the nuclei of koilocytes (Fig 7.2c, 7.2d). There was a band of heavy staining of the outermost edge of the stratum comeum with the P2Xs receptor antibody (Fig 7.2a), which was still present in the no primary antibody control (Fig 7.2e), and therefore was non-specific staining. There was no staining in the no primary control for the P2X? receptor antibody.

226 P2Xs and P2X? receptors in paraffin sections of raft cultures of normal human kératinocytes and of CIN612 (HPV 31) cells.

P2X5 immunoreactivity was present throughout all the layers of the raft cultures of normal human foreskin kératinocytes (Fig 7.3a), where the staining was confined largely to the cell membranes and the cytoplasm. P2X? immunoreactivity was present in the raft cultures of normal human foreskin kératinocytes, staining weakly within the uppermost layer (rudimentary stratum comeum) (Fig 7.3b). The P2X? receptor staining was not as strikingly positive as with the paraffin section of normal skin (Fig 7.1b).

P2X5 immunoreactivity was present in the CIN 612 (HPV 31) raft kératinocytes (Fig 7.3c), where the staining was seen within all layers of the raft. P2X? immunoreactivity was present in the CIN 612 raft (Fig 7.3d) and was associated with the cell cytoplasm in the HPV infected cells. At higher power, the uppermost layers are highly disorganised, with nucleated cells at the surface of the raft (Fig 7.3e, 7.3f). There was positive staining in the cytoplasm of mitotic cells within the raft for both P2Xs (Fig 7.3e) and P2X? (Fig 7.3f) receptors. There was no staining in the no primary control for both the P2Xs and the P2X? receptor antibody.

227 DISCUSSION

This study showed the first direct evidence for the expression of P2Xs and P2Xv, receptors in human warts, using immunohistochemistry. Studies on normal human epidermis and functional studies on primary keratinocyte cultures (see Chapter 2) have suggested that P2Xg receptors are likely to be involved in keratinocyte differentiation and P2Xv receptors are likely to be part of the machinery of end stage terminal differentiation of kératinocytes. 2’-& 3’-0-(4-Benzoyl-benzoyl) adenosine 5’- triphosphate (BzATP), a potent P2Xv receptor agonist, was shown to cause a significant decrease in cell number via a direct effect on P2X? receptors, which are also known to be involved in mediating apoptosis (Zheng et al. 1991; Ferrari et al. 1996). In normal skin, P2Xs receptors were found in the basal layer, stratum spinosum and weakly in the stratum granulosum. There was little or no P2Xs receptor staining in the stratum comeum, apart from some staining artefact. In the raft cultures of normal human foreskin kératinocytes, P2Xg receptors were found throughout all layers, from the basal layer to the most differentiated layer. Warts arise because human papillomavimses infect the basal keratinocyte of the epidermis, presumably through disruptions of the skin or mucosal surface (Cook and McKee 1989). The virus remains latent in basal cells as a circular episome. As kératinocytes differentiate and migrate to the surface, the vims is triggered to undergo replication and maturation. Hybridisation studies in situ of HPV lesions have shown that viral DNA synthesis occurs in the skin in the superficial stratum spinosum and full vims assembly with capsid production occurs in the stratum granulosum. In warts there is a prominent granular cell layer, within which there are vacuolated cells called koilocytes, characteristic of HPV infection. The process of vims replication alters the character of the epidermis, resulting in cutaneous or mucosal excrescences known as warts. In warts, P2Xg receptor staining was increased compared to that in normal skin. There were few P2Xs receptor positive cells in the basal layer, most of the positively stained cells being in the suprabasal layers. P2Xs receptors were found in the nuclei of koilocytes, indicating that these receptors were present in an abnormal location in vims infected cells, because they were usually found in the cytoplasm and cell membrane. In the CIN 612 (HPV 31) raft cultures, P2Xs receptors were found in all cell layers and the level of staining was more intense than in the normal keratinocyte raft cultures. There is evidence from other tissues regarding the role of P2Xs receptors. P2Xs receptors have

228 been implicated in the regulation of osteoblastic differentiation and proliferation (Hoebertz et al. 2000), and in triggering the differentiation of skeletal muscle satellite cells (Ryten et al. 2002). In fetal rat skeletal muscle, P2Xs receptors are sequentially expressed during development (Ryten et al. 2001). In normal human skin, P2X? receptor immunoreactivity was solely associated with cells and cell fragments within the stratum comeum (see Chapter 2). In the raft cultures of normal human foreskin kératinocytes, P2X? receptor staining was very weak compared to normal skin. This may be due to the phenomenon of incomplete differentiation that occurs in raft cultures, thought to be associated with the presence of retinoids in the medium (Kopan et al. 1987). In warts, P2X? immunoreactivity was associated with hyperkeratotic areas of the stratum comeum, as well as in nuclei of koilocytes in the suprabasal layers. The nuclei that were positive for P2X? receptors were not normal: nuclei were shmnken, with much more intense, pink P2X? receptor staining. In the CIN 612 raft cultures, P2X? immunoreactivity was positive in both the cell cytoplasm and in the nucleus of HPV infected cells. The P2X? receptor is a bifunctional molecule, that can be triggered to act as a channel permeable to small cations, or on prolonged stimulation form a cytolytic pore permeable to large hydrophilic molecules up to 900 Da (Surprenant et al. 1996). The opening of this pore results in the increase in intracellular cytosolic free calcium ions and the induction of cell death (Zheng et al. 1991; Ferrari et al. 1996). It is possible that the presence of P2X? receptors in the nucleus of HPV infected cells indicates a severe dismption of the cellular machinery. It might be possible to use P2X? receptor agonists to trigger apoptosis in these virally infected cells. P2X? receptors are also found on dendritic cells, macrophages and microglial cells, where extracellular ATP can trigger apoptosis via these receptors and there is increasing evidence that this process is dependent on the caspase signalling cascade (Coutinho-Silva et al. 1999; Ferrari et al. 1999). In summary, P2Xs and P2X? receptors may provide a useful focus for more research into new treatment modalities for warts and squamous cell carcinomas because these receptors can induce cell differentiation as well as cell death. Vegetative reproduction of HPV particles can only take place in highly differentiated kératinocytes. Raft organotypic cultures of both normal human kératinocytes and HPV infected cells could prove to be a useful tool for further study of these receptors in vitro.

229 FIGURE LEGENDS

Figure 7.1: Expression of P2Xg and P2X? receptors in paraffin sections of normal human skin. Nuclei were counterstained blue with haematoxylin. (a) In normal skin, P2Xs immunoreactivity (brown) was found in basal kératinocytes (SB) (although this was not easy to distinguish from melanin), and in the stratum spinosum (SS), and in very few cells in the stratum granulosum (SG). P2Xs receptor staining was absent from the stratum comeum (SC), apart from at the outer edge. P2Xg receptor staining was confined largely to the cell membranes and the cytoplasm, and occasionally in the nucleus. Scale bar 25pm. (b) P2X? immunoreactivity (pink) was present in the epidermis of all normal skin samples, and was associated with cells and cell fragments (arrow) in the stratum comeum (SC). Note the brown melanin in the stratum basale (SB). Scale bar 25pm. (c) There was residual staining of the outermost edge of the stratum comeum (arrow) with the P2Xg receptor antibody no primary control, and therefore this was non-specific staining. Note the brown melanin in the stratum basale (SB). Scale bar 25pm. (d) There was no staining in the no primary control for the P2X? receptor antibody Scale bar 25pm.

230 231 Figure 7.2; Expression of P2Xs and P2X? receptors in paraffin sections of human warts. Nuclei were counterstained blue with haematoxylin. (a) Low power view of P2Xs immunoreactivity (brown) in the wart. P2Xs receptors were present within the kératinocytes of the wart. There was marked hyperkeratosis (H), which was negative for P2Xs receptors, although areas of parakeratosis were positive (arrow). At the outer edge of the stratum comeum there was a band of heavy staining (asterisk). P2Xs receptors were also found in the inflammatory cell infiltrate (I) above the stratum comeum (SC). There was a prominent stratum granulosum (SG), within which cells (koilocytes) showed typical cytoplasmic vacuolation (arrowheads). Scale bar 100pm. (b) Low power view of P2X? immunoreactivity (pink) in the wart. P2X? receptors were strongly present within the hyperkeratotic (H) areas of the stratum comeum (SC), but not in areas of parakeratosis (arrow). P2X? receptors were weakly present in the wart kératinocytes, and mainly found in the nucleus. P2X? receptors were also weakly found in the inflammatory cell infiltrate (I) above the stratum comeum. Scale bar 100pm. (c) High power view of P2Xs immunoreactivity (brown) in wart kératinocytes. There were a few positive cells in the stratum basale (SB), but most of the positively stained cells were in the suprabasal layers. Koilocytes showed P2Xg receptor staining in the nucleus (arrowheads). Scale bar 50pm. (d) P2X? immunoreactivity (pink) was present in the suprabasal layers of the wart, in either large, flat nuclei with an obvious nuclear membrane (arrow), or in koilocytes, where the receptor was prominent in shrunken, pynotic nuclei, (arrowheads). P 2X7 receptors were not found in the stratum basale (SB) of the wart. Scale bar 50pm. (e) There was residual staining of the outermost edge of the stratum comeum (asterisk) with the P2Xs receptor antibody no primary control, and therefore this was non-specific staining. Scale bar 100pm. (f) There was no staining in kératinocytes of the wart with the P2Xg receptor antibody no primary control. There was some melanin in the stratum basale (SB) of the wart. Scale bar 50pm.

232 ,#r#

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233 Figure 7.3: Expression of P2Xs and P2Xy receptors in paraffin sections of raft cultures of normal human kératinocytes and of CIN612 (HPV 31) cells. Nuclei were counterstained blue with haematoxylin.

(a) P2X5 immunoreactivity (brown) was present throughout all layers of the raft cultures of normal human foreskin kératinocytes, where the staining was confined largely to the cell membranes and the cytoplasm. The raft culture was supported on a collagen matrix (C). Scale bar 25pm. (b) P2X? immunoreactivity (pink) was present in the raft cultures of normal human foreskin kératinocytes, staining weakly within the uppermost layer (arrows). Scale bar 25pm. (c) P2Xs immunoreactivity (brown) was present in the CIN

612 (HPV 31) raft kératinocytes, staining all layers of the raft culture. Scale bar 50pm.

(d) P2X7 immunoreactivity (pink) was present in the CIN 612 raft and was associated with the cell cytoplasm and nucleus (arrow). Scale bar 50pm. (e, f) High power views of CIN 612 (HPV 31) raft cultures: the uppermost layers are highly disorganised, with nucleated cells at the surface of the raft (arrows). There was also positive staining in the cytoplasm of mitotic cells (double arrows) within the raft for both (e) P2Xg receptors (brown) Scale bar 25pm. and (f) P2X? receptors (pink). Scale bar 25pm.

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235 CHAPTER 8 GENERAL DISCUSSION

236 The work presented in this thesis has focused on purinergic signalling in human epidermal kératinocytes in both normal adult and fetal epidermis, hair follicles, wound healing, non-melanoma skin cancers and cutaneous warts. A detailed discussion of the results was provided at the end of each experimental chapter. In this general discussion I would like to summarise the major findings and discuss some advantages and disadvantages of the techniques used; discuss long term trophic roles of purine and pyrimidines nucleotides with respect to the skin; examine the contributing factors in wound healing and relate them to purinergic signalling; discuss purinergic signalling and therapeutic implications for skin diseases and other pathological conditions and explore future directions for therapeutic applications.

1. Major findings of this thesis

This project was based on work in rat epidermis which used immunohistochemistry to localise the presence of P2Xs and P2X? receptors on epidermal kératinocytes and in hair follicles (Groschel-Stewart et al. 1999a). This work suggested functional roles for these purinergic receptors in the regulation of proliferation, differentiation and cell death in kératinocytes. Contradictory effects of purine nucleotides on keratinocyte proliferation have previously been reported. Adenosine triphosphate (ATP) at concentrations of 1-lOOpM inhibited terminal differentiation and stimulated thymidine incorporation in keratinocyte cultures (Pillai and Bickle 1992). Other investigators suggested that adenosine, deoxyadenosine and adenine nucleotides inhibited growth in both human and porcine kératinocytes (Harper et al. 1974; Flaxman and Harper 1975; Taylor et al. 1980; lizuka et al. 1984). Another study established that proliferation of keratinocyte cultures was inhibited by exogenous adenosine, AMP, ADP, ATP and ATPyS (Cook et al. 1995). At that time it was not known which purinergic receptor subtypes were present on human epidermal kératinocytes, nor which receptor subtypes might be mediating the functional effects of

ATP and adenosine. P 2Y2 receptor mRNA was then found in the basal layer of normal epidermis and DTP, a P 2 Y2 receptor agonist, stimulated keratinocyte proliferation (Dixon et al. 1999). Human kératinocytes have also been shown to express adenosine

A2b receptor mRNA (Brown et al. 2000), although these receptors did not mediate the anti-proliferative effect of adenosine on human keratinocyte cultures. In the first experimental chapter (Chapter 2), the expression of P2 receptors was studied in frozen sections of human skin using immunohistochemistry and functional

237 experiments on primary cultures of human epidermal kératinocytes were performed.

P2Xs, P2X7, P2Yi and P2 Y2 receptors were expressed in spatially distinct zones of the human epidermis. Functional studies were performed on primary cultures of human kératinocytes and on explanted rat skin, where different P2 receptor subtype agonists and antagonists were applied to cultured kératinocytes or injected subcutaneously into the skin, respectively. This showed that P2 receptors had different functional roles in the human epidermis, with P2Yi and P 2Y2 receptors controlling proliferation while P2Xs and P2X? receptors controlled early differentiation and terminal differentiation of kératinocytes respectively. In Chapter 3, the expression of P2 receptors was studied in frozen sections of human anagen hair follicles. P2 receptors were expressed in spatially distinct zones of the anagen hair follicle and co-localisation experiments suggested different functional roles for these receptors. P2Yi receptors were found in the outer root sheath and bulb;

P2Xs receptors were found in the inner and outer root sheaths and medulla; P 2 Y2 receptors were found in living cells at the edge of the cortex/medulla and P2X? receptors were not expressed. Co-localisation experiments suggested different functional roles for these receptors: P2Yi receptors were associated with bulb and outer root sheath keratinocyte proliferation; P2Xs receptors were associated with differentiation of cells of the medulla and inner root sheath; P 2 Y2 receptors were associated with early differentiated cells in the cortex/medulla which contribute to the formation of the hair shaft. In Chapter 4, the expression of P2 receptors was studied in frozen sections of human fetal epidermis. Each of the four receptors was expressed in spatially distinct zones of the developing epidermis: P2Yi receptors were found in the basal layer, P2Xs receptors were predominantly in the basal and intermediate layers, both P 2 Y2 and P2X? receptors were in the periderm. Co-localisation experiments suggested different functional roles for these receptors: P2Yi receptors in fetal keratinocyte proliferation;

P2X5 receptors in fetal keratinocyte differentiation; and P2X? receptors in apoptosis of periderm cells. The role of P 2 Y2 receptors in periderm cells is not known. So in summary, in normal tissues, P2Y% receptors appear to have role in proliferation, P2Xs receptors appear to be associated with differentiation and P2X? receptors appear to be involved in terminal differentiation, a specialised form of apoptosis. In adult epidermis, P 2Y2 receptors are found in basal cells which are also positive for markers of proliferation, and the receptors can be stimulated to cause significant proliferation with UTP. However in fetal epidermis, P 2Y2 receptors are only

238 present in the transient, fetal specific cell layer, the periderm. In anagen hair follicles,

P2Y2 receptors were expressed on early differentiated hair kératinocytes in the cortex that contribute to the formation of the hair shaft. In both fetal epidermis and in hair follicles, P 2 Y2 receptors did not co-localise with markers of proliferation. So, the role of

P2Y2 receptors may be more complex than just simple proliferation. It is possible that in the anagen hair follicle, P 2 Y2 receptors may represent a type of cell-survival factor, which keeps a cell able to proliferate and stops it from completing differentiation. It would be of interest to study when P 2 Y2 receptors become expressed in the basal layer in fetal epidermis. I studied fetal skin at 9-11 weeks gestation - it would be useful to obtain specimens from earlier and later points in gestation, and now we have developed a method whereby purinergic receptors can be immunolocalised in paraffin section, it may be possible to use archive rather than fresh frozen tissue. P 2 Y2 receptors on periderm cells could be involved in chloride and fluid secretion into the amniotic fluid.

P2 Y2 receptors are involved in ion and fluid secretion in lung and conjunctival epithelial cells (Yerxa 2001). Studies have suggested that non-keratinised fetal epidermis may have an osmoregulatory function and also that the fetal epidermis contributes to the formation of amniotic fluid in the first trimester and becomes the primary source in the second trimester (Holbrook 1991). Periderm cells have microvilli and surface blebs which increase the surface area of the plasma membrane exposed to the amniotic fluid, there are large numbers of membrane bound vesicles adjacent to the plasma membrane and tight junctions join adjacent periderm cells. This suggests that the periderm cell layer may have potential for fluid and ion transport (Holbrook 1991). Further work would have to be done to examine this hypothesis. Future directions for hair follicle experiments could include examining the P2 receptor subtype expression in all the different phases of the hair cycle, including catagen, where the lower portion of the hair follicle undergoes programmed cell death (Cotsarelis 1997). Chapter 5 investigated the changes in the expression of purinergic receptors in the regenerating rat epidermis during normal wound healing, in denervated wounds, and in denervated wounds treated with nerve growth factor (NGF), where wound healing rates were normalised. Changes in the level of receptor expression were quantified using optical density measurements of immunopositive epidermal kératinocytes. P2Xs,

P2X7, P2Yi and P2 Y2 receptor expression in the epidermis was altered during wound healing. P2Yi receptor expression was significantly increased in kératinocytes of the regenerating epidermis of denervated wounds but P 2 Y2 receptor expression was significantly decreased. NGF treatment enhanced P2Xs and P2Y1 receptor expression in

239 epidermal kératinocytes of innervated wounds. NGF treatment ‘normalised’ the expression of P2Yi and P 2Y2 receptors in kératinocytes from denervated wounds. P2X? receptors were absent in all experimental wound healing preparations.

SCCs. P2Yi receptors were only expressed in the stroma surrounding tumours. P 2Y4 receptors were found in BCCs but not in SCCs. The P2X? receptor agonist benzoylbenzoyl-ATP and high concentrations of ATP (1000-5000|xM) caused a significant reduction in A431 cell number (P<0.001), while the P 2 Y2 receptor agonist UTP caused a significant amount of proliferation (P<0.001). I have demonstrated that non-melanoma skin cancers express functional purinergic receptors and that P2X? receptor agonists significantly reduce cell numbers in vitro. Chapter 7 explored purinergic receptor staining in paraffin sections of cutaneous warts and in raft organotypic cultures of human foreskin kératinocytes and a human papilloma virus infected cell line, CIN-612, a model of kératinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of

P2X5 receptors. A similar pattern was seen in the CIN-612 raft cultures. Both P2Xg and

P2X7 receptors were found in the nuclei of koilocytes, abnormal kératinocytes characteristic of human papillomavirus infection. P2Xs and P2X? receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death. Vegetative reproduction of HPV particles can only take place in highly differentiated kératinocytes. Raft cultures of both normal human kératinocytes and HPV infected cells could prove to be a useful tool for further study of the role of these receptors. Immunohistochemistry was a good technique to show the tissue distribution of different P2 receptor subtypes, which were expressed in spatially distinct zones of the epidermis. P 2X 1.7 receptor antibodies were not commercially available and were kindly

240 provided by Roche Bioscience (Palo Alto, California, USA). P2Yi, P 2 Y2 and P2 Y4 receptor antibodies were available from Alomone Labs (Jerusalem, Israel). The immunogens used for production of polyclonal P2X antibodies were synthetic peptides corresponding to 15 receptor-type-specific amino acids (AA) in the intracellular C- termini of the cloned rat P2X receptors. These antibodies were used in all experiments because in general the ‘human’ antibodies provided by Roche Bioscience did not give as good staining - there was much background staining and there was also non-specific nuclear staining, which was not seen with the ‘rat’ antibodies. The reactivity of the P2Yi receptor antibody had been confirmed in rat and human tissues, whereas the reactivity of the P2 Y2 and P2 Y4 receptor antibodies used had only been confirmed in rat tissues, although the anti-P 2 Y2 receptor had 17/19 residues identical with the human

P2 Y2 receptor peptide. Double-labelling techniques were a useful method to demonstrate the relationships between P2 receptor subtype expression in the epidermis and markers of proliferation, differentiation and cell death, which are found in different zones of the epidermis. Another method to examine the expression of P2 receptor subtypes in kératinocytes at different stages of differentiation would be to take monocultures of kératinocytes grown in low calcium medium and induce differentiation by adding calcium to the medium and examining the expression of receptor subtypes at different time points, by either immunostaining or Western blotting. The induction of differentiation with this method takes about 6 hours and is complete by 24 hours (Hodivala and Watt 1994). In both primary human keratinocyte cultures and in a squamous cell carcinoma cell line, A431, P2 Y2 receptors could be stimulated with UTP to cause an increase in cell number, and P2X? receptors could be stimulated with BzATP to cause a significant decrease in cell number. It would be important to confirm that the increase in cell number stimulated by UTP was due to proliferation, rather than lack of cell death. Future experiments could establish this via either tritiated thymidine uptake or detection of 5-bromo-2’-deoxyuridine (BrdU) incorporation in DNA synthesising cells. A fluorescence activated cell sorter (FACS) could sort cells into proliferating and non proliferating compartments. BrdU is an analogue of thymidine and will be taken up into the DNA of cycling cells. To detect this, DNA can be unwound (by using acid, alkali or enzyme) and then an antibody against BrdU used. In this way, Gl, S and 02 cells could be separated. BrdU-positive cells would equate to S phase.

241 It would be useful to confirm that high dose ATP and BzATP are causing cell death rather than inhibition of proliferation in the primary keratinocyte cultures and in the A431 cell line. There was a 40% (Fig 2.7) and a 70% (Fig 6.9) decrease in cell number respectively at 48 hours after a single application of 500pM BzATP, and looking at the cells treated with high dose ATP, they were ‘rounded up’ and abnormal (Fig 6.7). It seems that the cancer cells are more sensitive to the effects of BzATP than normal kératinocytes, and this could have important implications for toxicity and side effect profiles. Apart from its agonist effect on P2X? receptors, BzATP also has potent agonist actions on P2Xi and P2Xg receptors, but these were shown not to be expressed in the epidermis. Future experiments could use flow cytometry and FACS analysis, which is the most rapid and precise method to sort apoptotic and healthy cells in culture, and can be used to separate cells from replicative and differentiating compartments. Because cells remain intact after sorting by FACS, markers of differentiation and apoptosis can be readily determined in sorted cells. In FACS, forward scatter (light intensity) correlates directly with cell size in many cell types, and this parameter has been used successfully to categorize cultured kératinocytes according to their status of differentiation (Jones and Watt 1993). Therefore it would be possible to examine the effects of stimulating cultured kératinocytes with lower doses of ATP or ATPyS, a potent agonist at P2Xs receptors, and measuring the change in the proportion of differentiating cells by using FACS analysis. This could confirm whether P 2X5 receptors caused keratinocyte differentiation and if this led to a reduction in keratinocyte proliferation. Flow cytomtery can also be used to count cells (Brando et al.

2000). If BzATP and high dose ATP did induce cell death, it seems likely that P2X? receptors in cutaneous warts could be also stimulated with BzATP to cause cell death. Future experiments to examine this could use the raft organotypic cultures of CIN-612 cells treated with high dose ATP or BzATP and then sectioned and stained for either TUNEL or active caspase-3. Future experiments to further investigate the anti-cancer activity of P2 receptor agonists and antagonists could include using an in vivo animal model, whereby A431 cells are injected into nude mice followed by intraperitoneal injections of BzATP. Tumour growth could be assessed by measuring various outcomes - tumour size, weight, and number of métastasés. Alternatively, human squamous cell carcinomas could be excised from patients with a cuff of normal tissue and transplanted/grafted

242 onto the backs of nude mice and e.g. BzATP could be injected subcutaneously into the tumour or the drug could be applied topically as a cream. Other purinergic receptor agonists and antagonists were also investigated in cultured kératinocytes and in A431 cells. In this study, the effect of ATP was more potent than that of adenosine in cultured kératinocytes. The effect of adenosine was not blocked by the adenosine (A 1/A2) receptor antagonist, 8 -(p-sulfophenyl)theophylline, and dipyridamole did not have a significant effect on the response to adenosine, suggesting that Ai and A 2 adenosine receptors did not mediate the response. An intracellular site of action of adenosine has previously been proposed (Brown et al.

2000). In the present study, the response to ATP was not significantly inhibited by 8 -(p- sulfophenyl)theophylline and dipyridamole had no significant effect in both cultured kératinocytes and A431 cells, so it seemed that the main trophic agent was ATP working on P2 receptors. While adenosine had an effect, it was minor and obscured by the effect of ATP. A decision was made to focus further functional studies on P2 receptor agonist and antagonists. The P2X? receptor antagonists KN-62 (Humphreys et al. 1998) and Coomassie Brilliant Blue G (Jiang et al. 2000) were investigated and these drugs were discarded. KN-62 had no effect on A431 cells, although some block of the effect of BzATP was achieved in kératinocytes. Coomassie Brilliant Blue G did not block the effect of BzATP in either kératinocytes or A431 cells. The P2Yi receptor antagonist, MRS2179 (King et al. 2000) was also investigated and discarded because it had its own proliferative effects on both cell types and did not block the effect of 2MeSADP. The non-selective P2 antagonist, PPADS (Khakh et al. 2000) was also an ineffective antagonist in both cell types. Suramin had a significant proliferative effect of its own on A431 cells, when administered at lOOpM (P<0.001).

2. Long term trophic roles of purines and pyrimidines

Studies on purinergic signalling have focussed largely on short term actions of extracellular nucleotides and nucleosides in neurotransmission, secretion, muscle contraction, regulation of vascular tone and immune cell function (Bumstock 1997). There is growing recognition that purinergic receptors have major long-term trophic actions in development, regeneration and in pathophysiological conditions (Abbracchio and Bumstock 1998; Bumstock 2002a). Previous studies have shown that there are trophic actions of extracellular nucleotides and nucleosides might also act as trophic factors in both the central and

243 peripheral nervous systems (Neary et al. 1996). Specific extracellular receptor subtypes for these compounds are expressed on neurons, glial and endothelial cells, where they mediate strikingly different effects. These range from induction of cell differentiation and apoptosis, mitogenesis and morphogenetic changes, to stimulation of synthesis or release, or both, of cytokines and neurotrophic factors, both under physiological and pathological conditions. Nucleotides and nucleosides might be involved in the regulation of development and plasticity of the nervous system, and in the pathophysiology of neurodegenerative disorders. Receptors for nucleotides and nucleosides could represent a novel target for the development of therapeutic strategies to treat incurable diseases of the nervous system, including trauma, and ischaemia associated neurodegeneration, demyelinating and ageing associated cognitive disorders. Purinergic receptors have trophic roles in the migration, proliferation and cell death of vascular smooth muscle and endothelial cells (Bumstock 2002a). The mitogen- activated protein kinase (MAPK) second-messenger cascade is implicated in proliferation of vascular smooth muscle and endothelial cells, although details of the precise intracellular pathways involved still remain to be determined. Synergistic actions of purines and pyrimidines with growth factors occur in promoting cell proliferation. There is evidence for the release of ATP from endothelial cells, platelets, and sympathetic nerves as well as from damaged cells in atherosclerosis, hypertension, restenosis, and ischaemia. There is also evidence that vascular smooth muscle and endothelial cells proliferate in these pathological conditions. In a study of porcine aortic endothelial cells (von Albertini et al. 1998), extracellular ATP and ADP, probably acting through P2X? receptors, were shown to activate the nuclear factor kappaB (NF-k B) family of gene-regulatory proteins which play important roles in the induction of cell death (Baichwal and Baeuerle 1997).

Extracellular ATP has also been shown to activate NF-k B in microglial cells through the P2Xv receptor (Ferrari et al. 1997b). P2X? receptors have also been shown to have a role in triggering cell death in immune cells (see Chapter 1) and the anti-cancer effects of ATP may also be mediated via P2X? receptors. It has been proposed that P2Xs receptor have a trophic role in skeletal muscle differentiation (Ryten et al. 2002). P2Xg receptors are present on satellite cells and ATP activation of a P2X receptor inhibited proliferation and stimulated expression of markers of muscle cell differentiation, including myogenin, p 2 1 , and myosin heavy chain, and increased the rate of myotube formation. ATP application also resulted in a

244 significant and rapid increase in the phosphorylation of MAPKs, particularly p38. Inhibition of p38 activity prevented the effect of ATP on cell number. This thesis set out to study the trophic effects of ATP on epidermal kératinocytes. It seems that purinergic signalling may represent part of a fundamental mechanism found in many different cell systems, which is involved in the regulation of cell proliferation, differentiation and cell death.

I have shown trophic roles for P2Yi and P 2Y2 receptors in proliferation in normal epidermis, confirmed by functional studies in primary keratinocyte culture.

2MeSADP and UTP, the P2Yi and P2Y2 receptor subtype agonists respectively, also caused an increase in epidermal thickness after subcutaneous injection in rat skin. P2Y 1 receptors also appear to have a proliferative role in hair follicle and in fetal epidermis. In basal cell and squamous cell carcinomas, P2Y% receptors were not expressed on tumour cells and 2MeSADP caused a non-significant reduction in A431 cell number.

However, P 2 Y2 receptors were expressed on these non-melanoma skin cancers and UTP application caused a significant increase in A431 cell number. This suggests that the trophic roles of P2Y% and P 2 Y2 receptors with respect to proliferation are complex and also vary with cell type. A trophic role for P2Xs receptors has been implicated by double labelling studies with markers of differentiation in normal skin, hair follicles, fetal epidermis, non-melanoma skin cancers and warts. ATPyS, a potent agonist of P2Xs receptors, caused a decrease in both keratinocyte and A431 cell number, which could be because cells are lost from the cell cycle by being made to differentiate via activation of P2Xs receptors. P2Xs receptors were up-regulated in the stratum granulosum in warts, where koilocytes, the cell type indicative of HPV infection, are found. Vegetative reproduction of HPV particles can only take place in highly differentiated cells. A trophic role for P2X? receptors in terminal differentiation, a specialised form of physiological cell death, was shown in adult and fetal epidermis. P2X? receptors, are known to be involved in mediating apoptosis in other cell types. BzATP, a potent agonist of P2X? receptors, and high concentrations of ATP decreased both keratinocyte and A431 cell numbers via a direct effect on P2X? receptors, although cancer cells were more sensitive to the effects of BzATP than normal cells in culture. Extracellular ATP has also been shown to activate NF-k B in microglial cells through the P2X? receptor

(Ferrari et al. 1997b). Depending on the cell type, NF-k B plays either a pro-apoptotic or anti-apoptotic role (Baichwal and Baeuerle 1997). In the suprabasal layers of the epidermis, NF-k B translocates from the cytoplasm to the nucleus and induces target

245 gene expression. In light of its potent role in regulating apoptotic cell death in other tissues, NF-k B activation in these cells suggests that this transcription factor regulates cell death during terminal differentiation. P2X? receptors are also expressed in the terminally differentiated layers of the epidermis (see Chapter 2). However, NF-k B has been found to have a role in preventing premature and abnormal cell death in epidermal cells committed to undergoing terminal differentiation. It seems that NF-k B function is necessary for normal spatial control of cell death in the epidermis, and, in the epidermis, such cell death normally proceeds via a distinct pathway that is resistant to NF-k B and its anti-apoptotic target effector genes (Seitz et al. 2000). NF-k B may not therefore have a direct role in triggering physiological cell death in the epidermis, but rather a role in preventing premature apoptosis, so perhaps its main role is to promote keratinocyte viability during the process of outward migration and terminal differentiation until entry into the specialised epidermal cell death pathway occurs through other mechanisms. The relationship between NF-k B and P2Xv receptors within the epidermis therefore warrants further study.

3. Purinergic signalling in wound healing

While in Chapter 5 wound healing was discussed in terms of purinergic signalling and epidermal regeneration and NGF, I will now discuss the relevance of purinergic signalling to the process of wound healing in broader terms. A role for purines and pyrimidines in wound healing has previously been suggested (Abbracchio and Bumstock 1998). Wound healing involves the co-ordinated activity of inflammatory, vascular, connective tissue and epithelial cells. All of these components need an extracellular matrix to facilitate this. Skin wounds heal by the formation of an epithelialised scars, rather than by regeneration of a tme full thickness tissue. The aims of wound healing research are to find ways to minimise scar formation and to accelerate healing time. Purinergic receptors are found in monocytes, macrophages, mast cells, neutrophils, platelets, erythrocytes, vascular smooth muscle and endothelial cells, dermal fibroblasts and epidermal kératinocytes. Purinergic signalling is involved in haemostasis, which is important just after a cutaneous wound is made. ADP is a potent platelet recruiting factor and induces platelet aggregation via interaction with two P2 platelet receptors, the P2Yi receptor, that is involved in shape changes and transient platelet aggregation, and the P 2 Yi2 receptor

246 that mediates degranulation and sustained platelet aggregation (Hollopeter et al. 2001; Cachet 2001). ATP is a competitive ADP antagonist at platelet P2Y receptors and

stimulates the production of PGI 2 and NO, which can also inhibit platelet aggregation and act as vasodilators. Exogenous ATP acts to localise thrombus formation to areas of vascular damage, controlling the relationship between haemostasis, thrombosis and fibrinolysis (Bumstock and Williams 2000). The acute phase of wound healing involves acute inflammation. ATP and adenosine are released at sites of inflammation. ATP is involved in the development of inflammation through a combination of actions: release of histamine from mast cells, provoking production of prostaglandins, and the production and release of cytokines from immune cells (Bumstock 2002b). Purinergic receptors on immune cells are also involved in the killing of intracellular pathogens by inducing apoptosis of host macrophages, chemo-attraction and cell adhesion. In contrast, adenosine exerts anti inflammatory actions. Adenosine inhibits neutrophil rolling and adhesion to vascular

endothelium, decreases oxygen free radical production by neutrophils via A 2A receptor activation, and also exerts effects on endothelial cell permeability, reducing bradykinin

and histamine-induced vascular leakage via activation of Ai and A 2A receptors (Williams and Jarvis 2000). Wound healing also involves new vessel ingrowth (angiogenesis) into the wound healing matrix. Adenosine and ATP are involved in cell migration and proliferation during angiogenesis (Bumstock 2002a). Adenosine inhibits vascular

smooth muscle cell proliferation by A 2 receptor activation (Jonzon et al. 1985; Dubey et al. 1996). ATP and ADP stimulate DNA synthesis and cell proliferation in cultured porcine artery smooth muscle cells (Wang et al. 1992), and ATP is mitogenic in human vascular smooth muscle cells (Erlinge et al. 1994). UTP also has powerful mitogenic actions on vascular smooth muscle (Erlinge et al. 1995; Malam-Souley et al. 1996) and

it has been suggested that P 2 Y2 and P2 Y4 receptors might be implicated in this

(Bumstock 2002a). There is evidence for P2Yi, P 2 Y2 and P2 Y4 receptor subtypes on vascular endothelial cells, which are involved in mediating the release of NO, and causing subsequent vasodilatation (Bumstock 2002a). Endothelial cells release ATP (Pearson and Gordon 1979; Bodin et al. 1991), and it has been suggested that ATP released from vascular endothelial cells causes an autocrine mitogenic stimulation of endothelial cells (Bumstock 2002a). Adenosine can also stimulate vascular endothelial cell proliferation (Van Daele et al. 1992; Ziche et al. 1992; Ethier et al. 1993) and DNA

synthesis (Ethier and Dobson, Jr. 1997). Adenosine A 2A receptors have been shown to

247 mediate endothelial proliferation in human umbilical veins via activation of MAPK (Sexl et al. 1995, 1997). Adenosine also mediates vascular endothelial growth factor (VEGF) expression via Aza receptors in human retinal endothelial cells (Grant et al.

1999) and A 2B receptor antagonists inhibit proliferation of human retinal endothelial cells stimulated by the adenosine analogue NEC A (Grant et al. 2001). Adenosine may also act independently from adenosine receptors. In bovine aortic endothelial cells, the mitogenic action of adenosine was resistant to the adenosine receptor antagonist, 8- phenyltheophylline and was not mimicked by A% and A2 selective agonists (Van Daele et al. 1992). An intact nerve supply is important for normal wound healing. In denervated wounds, healing is delayed (Richards et al. 1997). Sympathetic nerves release ATP that can activate vascular smooth muscle (Erlinge et al. 1994) and endothelial cell (Bumstock 2002a) proliferation, to induce angiogenesis, and stimulate proliferation in epidermal kératinocytes (see Chapter 2) and fibroblasts (Wang et al. 1990) to promote scar formation.

Topically applied adenosine A% or A 2A receptor agonists promotes cutaneous wound healing in full thickness excisional wounds in mice and rats (Montesinos et al.

1997; Sun et al. 1999). Evidence from experiments performed on adenosine A 2A receptor knockout mice demonstrates that the A 2A receptor is the adenosine receptor subtype involved in wound healing and its function is to promote angiogenesis

(Montesinos et al. 2002). Adenosine A 2A receptor agonists also increased the rate of wound closure (Victor-Vega et al. 2002). Other groups have found that the adenosine

A2A receptor promotes collagen production by dermal fibroblasts (Chan et al. 2001). There has been no previous investigation of P2 receptor subtype expression in animal models of cutaneous wound healing, and the effect of ATP in cutaneous wound healing has not yet been examined. In the present study, the expression of P2 receptor subtypes in the regenerating epidermis in several different types of wounds in a rat model was examined. Normal wounds, denervated wounds (as a model of chronic wounds) and denervated wounds treated with NGF, where wound healing rates are normalised were examined. The level of expression of both P2Yi and P 2Y2 receptors was shown to be unchanged in the epidermis of control wound edges compared to normal skin. However, the distribution of the receptors within the epidermis was altered, with both receptors expressed in suprabasal as well as basal kératinocytes at the epidermal wound edge. This could represent part of the change of phenotype that kératinocytes undergo in order to become migratory during the wound healing process.

248 The expression of both P2Xs and P2Yi receptors was significantly increased (P<0.001) in kératinocytes of the regenerating epidermis of denervated wounds compared to control wounds, whereas the expression of P2Y% receptors was significantly decreased (P<0.001). In epidermal kératinocytes from NGF-treated control wounds there was a statistically significant increase (P<0.001) in P2Xs receptor expression compared to untreated control wounds. It seemed that an intact nerve supply was necessary to up- regulate the expression of P2Xs receptors with NGF, because there was no statistically significant change in the level of P2Xs receptor expression in kératinocytes of the regenerating epidermis of NGF-treated denervated wounds compared to untreated denervated wounds. NGF has a normalising effect on the rate of healing of denervated wounds (James et al. 2001) which is usually delayed (Richards et al. 1997). The expression of both P2Yi and P 2Y2 receptors was ‘normalised’ in kératinocytes of the regenerating epidermis of NGF-treated denervated wounds. During the proliferative phase of epidermal wound healing, keratinocyte proliferation is increased and apoptosis is reduced (Miadelets 1995; Nagata et al. 1999), so that the regenerating epidermis is thickened. P2X? receptors are strongly linked to apoptosis (Surprenant et al. 1996; Collo et al. 1997). This study showed that the expression of P2X? receptors in kératinocytes at the normal wound edge was reduced compared to normal epidermis and this suggested that the reduction in P2X? receptors was linked to the reduction in apoptosis in the healing epidermis. P2X? receptors were absent in both untreated and NGF-treated experimental wound healing preparations. Future directions for research could include, investigating the P2 purinergic receptor expression at more than one time point. In this study, tissue was harvested at day 4 after wounding, when the wounds were still open. Further work is needed to elucidate the functional role of these receptors in cutaneous wound healing. Future experiments that might be fruitful include: topical application of P2 purinergic receptor subtype agonists and antagonists to excisional wounds and evaluation of the rate of wound closure by, e.g. tracing wound outlines onto clear plastic sheets and measuring the wound area by digitising the tracings; and also evaluating the amount of cellular proliferation in the wound by, e.g. using in vivo incorporation of 5-bromo-2’- deoxyuridine (BrdU), followed by immunostaining of tissue sections to identify cells in S-phase (DNA synthesis phase). Studies in knockout mice could also be of interest. An adenosine A 2A knockout mouse has already been used for studies in cutaneous wound healing (Montesinos et al. 2002).

249 4. Purinergic signalling and therapeutic applications

There is increasing interest in the therapeutic potential of purinergic compounds in a wide range of diseases (Bumstock 2002b), in relation to both PI receptors (Müller and Stein 1996; Kaiser and Quinn 1999; Broadley 2000; Impagnatiello et al. 2001) and P2 receptors (Abbracchio and Bumstock 1998; Agteresch et al. 1999; Bumstock and Williams 2000). A number of purine-related compounds have been patented (Fischer 1999). In recent years, several clinical applications of ATP and adenosine have been reported. Therapeutic potential of purinergic signalling in general a) Nervous system Agonists and antagonists of PI and P2 receptors are being explored for a number of neurological conditions. For example, microinjection of ATP analogues into the prepiriform cortex induces generalised motor seizures (Knutsen and Murray 1997).

P2%2, P2X4 and P2Xô receptors are expressed in the prepiriform cortex, suggesting that a P2X receptor antagonist may have potential as a neuroleptic agent. In nervous tissue, trophic factors ensure neuronal viability and regeneration. Neuronal injury releases fibroblast growth factor, epidermal growth factor and platelet derived growth factor (Neary et al. 1996). In combination with these growth factors, ATP can stimulate astrocyte proliferation, contributing to the process of reactive astrogliosis, a hypertrophic/hyperplastic response associated with brain trauma, stroke, ischaemia, seizures and neurodegenerative disorders. Adenosine modulates long-term synaptic plasticity in the hippocampus, and it attenuates long-term potentiation. In accordance with the notion that synaptic plasticity is the basis for teaming and memory in different brain areas, adenosine correspondingly modulates behavior in various teaming and memory paradigms. Adenosine-related compounds might prove helpful in the treatment of memory disorders and the effects of adenosine on synaptic plasticity should be relevant for the enhancement of intellectual performance related to caffeine intake (de Mendonça and Ribeiro 2001). A 2A receptor antagonists are being investigated for the treatment of Parkinson’s disease (Williams 2001). ATP, given systemically, elicits pain responses and endogenous ATP may contribute to the pain associated with causalgia, reflex sympathetic dystrophy, angina, migraine and cancer pain (Bumstock 1996). P2Xs receptors are found on sensory neurons in trigeminal, nodose and dorsal root ganglia, and the terminals of these nociceptive neurons in the skin and visceral organs represent unique targets for novel

250 analgesic agents that function as P 2 X3 receptor antagonists. Non-specific P2 receptor antagonists, e.g. suramin, are anti-nociceptive, and P2Xg receptor-knockout mice have reduced nociceptive inflammatory responses (Cockayne et al. 2000). Intravenous adenosine has been shown to reduce neuropathic pain, hyperalgesia and ischaemic pain to a similar degree as morphine or ketamine (Segerdahl et al. 1994). Clinical trials have shown that peroperative infusion of adenosine reduced the requirement for volatile anaesthetic gas (isoflurane) and postoperative opioid use during surgery (Segerdahl et al. 1995, 1996,1997). b) Cardiovascular system Several studies have demonstrated the efficacy of intravenous injections of ATP and adenosine for the diagnosis and treatment of paroxysmal supraventricular tachycardias (for a review see Agteresch et al. (1999)). There have been some promising developments concerning purinergic antithrombotic drugs. Platelets express both P2X and P2Y receptors (Bumstock 2001a). Recent clinical trials, CAPRIE (CAPRIE Steering Committee 1996), and CURE (Yusuf et al. 2001) have provided clear evidence that the purinergic antithrombotic drugs clopidogrel and ticlopidine, which are antagonists of the platelet P 2 Yi2 receptor, reduce the risks of recurrent strokes and myocardial infarctions, especially when combined with aspirin. Patents have been lodged for the application of PI receptor subtype agonists and antagonists in myocardial ischaemia-reperfusion injury, cerebral ischaemia and stroke (Broadley 2000). ATP plays a significant co-transmitter role in sympathetic nerves supplying hypertensive blood vessels. Up-regulation of P2Xi and P 2Y2 receptor mRNA in the hearts of rats with congestive cardiac failure has been reported (Hou et al. 1999). Further therapeutic targets for P2 receptor agonists and antagonists include congestive cardiac failure, hypertension, stroke and angina. c) Pulmonary system

The P2 Y2 receptor coordinates mucociliary clearance in the upper and lower respiratory tract. This process can be regulated therapeutically by the local delivery of

P2 Y2 receptor agonists (Kellerman 2002). Studies have established that P 2Y2 receptors are found on each of the three principal cell types that line the airways: ciliated epithelial cells, goblet cells, and Type n alveloar cells. On activation of the P 2Y2 receptor on ciliated epithelial cells, salt and water are released from the cell, mucous secretions are hydrated, and ciliary beat frequency is increased. Activation of the P 2 Y2 receptor on goblet cells modulates the release of mucin. When the P 2 Y2 receptors on

251 Type n alveolar cells are activated, they release surfactant, a lubricating molecule that maintains the surface tension of the smallest peripheral airways and prevents their collapse. Cystic fibrosis is characterized by defective cystic fibrosis transmembrane regulator (CFTR) expression and function, associated with abnormal ion transport and mucociliary clearance, and clinical lung disease. Activation of P 2Y2 receptors has been found to both activate Cl" secretion and inhibit Na"*" absorption, which are abnormal in cystic fibrosis. Aerosol UTP inhaled in combination with a sodium channel blocker (amiloride) improved mucociliary clearance from the airways of cystic fibrosis patients to near normal (Bennett et al. 1996). Phase I clinical trials using purinergic compounds such as the P2 Y2 receptor agonist INS365 (Inspire Pharmaceuticals, Inc, North Carolina, USA) have confirmed the safety of this compound in cystic fibrosis patients

(Noone et al. 2001). The next-generation P 2Y2 receptor agonist INS37217 (Inspire Pharmaceuticals, Inc) is more metabolically stable, has an increased duration of improved mucociliary clearance and it may confer significant advantages over other

P2 Y2 agonists in the treatment of diseases such as cystic fibrosis, but this compound has so far only been tested in primate lung (Yerxa et al. 2002). Purinergic compounds are being explored to improve the clearance of secretions in chronic obstructive pulmonary disease (COPD) and for sputum expectoration in smokers (Yerxa 2001). Pulmonary hypertension can be a problem in patients with COPD. Intravenous ATP has been shown to exert pulmonary vasodilating effects, with significant decreases in mean pulmonary arterial pressure and pulmonary vascular resistance, without a change in mean systemic arterial pressure and systemic vascular resistance (Brook et al. 1994). The use of theophylline, an adenosine-receptor antagonist, as an anti-asthmatic agent has focussed attention on the development of novel PI receptor antagonists as asthma treatment (Biaggioni and Feoktistov 2001). ATP may have a direct role in asthma through its actions on bronchial innervation. Nucleotides trigger a reflex bronchoconstriction by activating a P2X receptor on vagal C fibres, and both ATP and UTP can potentiate IgE-mediated mast-cell histamine release, which involves P2Y receptors (Schulman et al. 1999). d) Ophthalmology

P2Y2 receptors are present on the ocular surface and conjunctiva of the eye, as well as on the retinal pigment epithelium. P 2 Y2 receptor agonists stimulate the secretion of salt, water, and mucus from the conjunctival mucosal surface (Li et al. 2001). This

252 mechanism of basal tear secretion forms the scientific basis for the clinical investigation of INS365 (Inspire Pharmaceuticals, Inc) as a tear film secretagogue for the treatment of dry eye disease (Fujihara et al. 2001, 2002). INS365 ophthalmic solution is currently in Phase n clinical trials in the USA (Yerxa 2001). In experimental models of retinal

detachment the P2 Y2 receptor agonist, INS37217 (Inspire Pharmaceuticals, Inc) was injected into the back of the eye and activated P2Y% receptors on the retinal pigmented epithelium, promoting fluid absorption in the subretinal space and thus facilitating retinal reattachment. This may provide a novel approach to the treatment of retinal detachment (Yerxa 2001).

e) Urogenital system ATP and adenosine have been used to protect the kidney from renal ischaemia- reperfusion injury, and are being explored for the treatment of chronic renal failure (Jackson 2001). Stimulation of the mucosal tissues of the female reproductive tract with

P2 Y2 receptor agonists results in hydrating and lubricating secretions. INS365 (Inspire Pharmaceuticals, Inc), as a vaginal gel, may be developed as a novel therapy for atrophic vaginitis associated with menopause. f) Musculoskeletal system The bisphosphonate clodronate, which is used in the treatment of Paget’s disease and tumour induced osteolysis, may act through osteoclast P2 receptors. A recent study has shown that nanomolar concentrations of ADP acting through P2Yi receptors activate osteoclast activity (Hoebertz et al. 2001). Modulation of P2 receptor function may have potential in the treatment of osteoporosis (Dixon and Sims 2000).

g) Oncology ATP and adenosine have also been shown to have a variety of effects on cancers: improvement of cancer cachexia, inhibition of tumour growth and a synergistic action on tumours when administered with chemotherapeutic drugs (this topic has already been extensively covered in Chapter 1). Therapeutic potential of purinergic signalling in skin disease There has been little work on therapeutic applications of purinergic signalling in the skin. This thesis has pioneered the investigation of the roles of purinergic receptors in the skin and laid the foundations for studies of therapeutic applications of purinergic receptors in thin skin, hair follicle biology, wound healing, non-melanoma skin cancer and warts.

253 Subcutaneous injection of P2 receptor agonists caused a change in the thickness of the epidermis in rat skin. This could have potential therapeutic applications, especially in patients with thin skin from chronic treatment with steroids. Future experiments to inject UTP, or more stable P 2Y2 receptor agonists currently being synthesised, into skin biopsies from patients with thin skin would examine whether an increase in epidermal thickness could be induced. If that was the case, then perhaps these compounds could be further developed into the form of a topically applied cream. The expression of P2 purinergic receptors in the anagen hair follicle may have therapeutic implications for the treatment of hair loss or excess hair. The expression of P2 receptors in different cell lineages of the anagen hair follicle could provide the prospect of engineering hair follicles in those with hair loss or excess hair, by using purinergic receptor agonists and antagonists. The expression of different P2 receptor subtypes has been shown to be altered during epidermal regeneration in wound healing. Specific agonist and antagonist drugs acting on purinergic receptors may lead to new approaches to wound re-epithelialisation. Non-melanoma skin cancers have been shown to contain functional purinergic receptors and P2X? receptor agonists can significantly reduce cell numbers. This may open a pathway for new treatment modalities in these important skin cancers.

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300 APPENDIX

301 Appendix - General Tissue Culture

Cell culture procedures were performed in sterile Class II laminar air flow hoods (LaminAir) from Heraeus Instruments. Cells were cultured in Heraeus incubators in a

humidified atmosphere at 37°C, enriched with 5% CO 2. Cells were centrifuged by use of Heraeus Labofuge. An Olympus CK2 inverted phase contrast microscope was used to examine live cells. Cells were counted using a Fuchs-Rosenthal haemocytometer. Dulbecco’s phosphate buffered saline (PBS) without Ca and Mg for tissue culture, Dulbecco’s Modified Eagles’ Medium (DMEM) and fetal calf serum (PCS) were obtained from Gibco Ltd. Other tissue culture products (trypsin, 0.05% ethylenediaminetetra-acetic acid (versene) and di-methyl sulfoxide (DMSO)) were obtained from Sigma Ltd. Greiner Labortechnik supplied culture flasks, universals, filters and other plastic-ware.

Feeder cells - 3T3 Swiss Mouse fibroblast cell line Stock cultures were grown on plastic in Dulbecco’s modification of Eagle’s Minimum Essential Medium (DMEM) supplemented with 10% fetal calf serum (ECS), penicillin/streptomycin and L-glutamine (but no HEPES). Cells were disaggregated just prior to confluency with trypsin/EDTA and seeded at 2 xlO^ cells per 10cm dish. Cultures were discarded after 25 passages and re-established from cryogenically stored stock cultures. Feeder cells were irradiated for 1 minute 50 seconds with a Cobalt^^ source to prevent them overgrowing when added to kératinocytes. 3T3 cells received 6000 rads of irradiation. A T75 flask of kératinocytes was seeded with 1ml of irradiated

feeder cells at 2 x 10^ cells/ml.

Normal human keratinocyte strains Human keratinocyte strains were initiated from skin from patients under age 30 I and were grown in the presence of irradiated 3T3 cells which were used as feeder layers (Rheinwald, 1980). The skin was harvested from operations where normal skin was

I discarded, for example, prominent ear corrections, breast reductions and abdominoplasties. Normal keratinocyte medium was made up as below. The medium was changed every 3 days and cells were passaged just prior to confluence and split 1:3. Cells at passage number 2 to 5 were used.

302 Normal Keratinocyte medium (as used for routine culture) This was made up as follows: • 500mls of F12/DMEM (NUT mix) (either 1:1 or 1:3) GIBCO Cat no. 21331-020 • SOmls FCS (Fetal calf serum) (GIBCO) • 5ml penicillin/streptomycin (xlOO) (10,000u/ml penicillin and 10,000ug/ml streptomycin GIBCO) • 5ml L-glutamine (xlOO) 200mM (GIBCO - cat no 25030-024) • 5ml RM+ medium (see below) • 3.5ml IM HEPES (Sigma) (0.2pm filter before adding) = buffer to keep cells at best pH (7.4) for growth.

Culture medium was made up into 500ml aliquots and stored at 4°C for a maximum of 6 weeks.

RM+ medium Add one vial of each of the following aliquoted growth factor stocks to lOmls of PBS/BSA: • EGF - epidermal growth factor (Sigma) lOOpl (1 mg/ml in PBS/BSA) • CT - cholera toxin (Sigma) 1ml (lOOpg/ml in PBS/BSA) • HC - hydrocortisone (Sigma) 1ml (4mg/ml in water) • I - insulin (Sigma) 1ml (50mg/ml in 0.05M HCl) • A - adenine (Sigma) 1ml (243.5mg/ml in water at pH 9.0 - add NaOH to adjust pH) • L - liothyronine (Sigma) lOOpl (1.3pg/ml - dissolve 13mg in 5mls of a 1:2 mix of IM HCl: ethanol, make up to 100ml with water and then dilute 20pl of this

1:100 in water to give 1.3pg/ml) • T - transferrin (Sigma) 1ml (50mg/ml in PBS/BSA) Make up to lOOmls with PBS/BSA. Filter sterilise and aliquot into 5mis and freeze - 20°C.

Low Calcium Keratinocyte Culture This allowed monoculture of kératinocytes without the need for 3T3 feeder cells, so that a proliferation assay could be performed on kératinocytes alone. Human keratinocyte strains were initiated as above and cultured with irradiated 3T3 feeder cells. Kératinocytes at passage 0 were disaggregated just prior to 70% confluency and

303 seeded into low calcium keratinocyte medium (see below) at passage 1. The cells were media changed at 24 hours after passaging to remove any dead kératinocytes and feeder cells that remained. At 48 hours after passaging, the cells were disaggregated with trypsin/EDTA and seeded into 96 well plates at 4 xlO^ cells per well for use in the proliferation assay.

Low calcium keratinocyte medium Make up as follows: • 200 mis of MCDB 153 medium (see below) • lOmls of chelated FCS (cFCS) (see below) • 2mls of RM+ medium (see above)

• 2mls of penicillin/streptomycin ( 10,0 0 0 u/ml penicillin and 10,0 0 0 ug/ml streptomycin GIBCO) • 2mls of L-glutamine (Sigma) Filter sterilise and store at 4 C.

MCDB 153 Medium

Measure out 850ml of H 2O (tissue culture-grade, source - Sigma, Cat# W 3500). Water temperature should be 15-20°C. While gently stirring the water, add the following: 1 litre pack of powdered MCDB 153 medium (source - Sigma, Cat# M 7403). 37.3mg L-Histidine (source - Sigma, Cat# H 9511) 98.4mg L-Isoleucine (source - Sigma, Cat# 17383) 13.4mg L-Methionine (source - Sigma, Cat# M 2893) 14.9mg L-Phenylalanine (source - Sigma, Cat# P 5030) 9.2mg L-Tryptophan (source - Sigma, Cat# T 0271) 19.55mg L-Tyrosine (source - Sigma, Cat# T 1145)

4.38mg CaCl 2.6H2 0 (source BDH, Cat# 100694A) (N.B. brings Ca^"^ levels up to 0.05mM). • 2g NaHCOs (source BDH, Cat# 102475W) (N.B. enough to buffer media at 5%

CO2 levels within incubator) Stir until dissolved. N.B. do not heat. While stirring, adjust the pH to 7.0 using either IN HCl or IN NaOH. Bring volume up to 1 litre and sterile filter (0.22p,m). Store for up to 3 months at 4°C.

304 Chelated Fetal Calf Serum This was used for low calcium cultures of kératinocytes to make calcium free serum.

• Weigh out 160g Chelex® 100 resin (200-400 mesh, sodium form, Source -

Biorad, cat # 142-2842) and wash in 2 litres of distilled H 2O with stirring for at least 1 hr at room temperature.

• Allow to settle for approximately 30 minutes and gently pour off distilled H 2O. • Add 500ml of FCS and stir for 2 hrs at room temperature. • Centrifuge at 3000 rpm for 10 minutes and gently pour off FCS. • Sterile filter FCS using 0.22pm filter and aliquot into lOmls and store at -20°C.

305