Proline rich acidic protein 1 in life and death 2

The expression of PRAP1 is significantly correlated with the degree of differentiation of gastrointestinal epithelial cells.. 83 Figure 3.18 PRAP1 expression is induced at mRNA level by

Assistant Professor Bernard Leung for his unfailing support, technical guidance, patience, and for providing his expertise in the work on SLE

Associate Professor Manuel Salto-Tellez of Pathology Department for his kind guidance and for providing his expertise in the histopathological analysis of human intestinal tissues

The wonderful present and past members of the Cancer Metastasis & Epigenetic Laboratory: Dr Liu Jian-Jun, for his stimulating discussions, valuable guidance and his contribution in the work on differentiation; Colyn, for her contributions in the work of bacteria binding, phagocytosis, pulldown assay, isolation and treatment of PBMCs; Jasmine, for her contribution in the antibacterial activity assay; Carol, Mirtha, Pui Nam, Yu Hong, Guo Hua, Guo Dong, Ganesan, Yun Tong, Jin Qiu, and Tamil for their advice and support Thank you all for your wonderful friendship and for making my stay in this laboratory truly enjoyable

Special thanks to the following people for their much appreciated and valuable input in proofreading this thesis: Koh Shiuan, Sufen, Seehee and Kah Weng

To all staff and students of the Department of Physiology, NUS, for their advice, support and friendship

To the Administration and Support Team of Physiology Department for their kind assistance and friendship Special thanks to Asha for her support in arranging all the meetings and many other administrative works

To past and present members of NUMI Confocal and Flow Cytometry Units, for their kind assistance

To my friends, Seehee, Suli and Jaws for their unfailing friendship and support throughout this course

Last but not least, to my parents and siblings, for their love, understanding, care and support throughout my study

And above all, all glory to God for His unfailing grace, mercy and love, and for granting me strength in Him to complete this course

SOLI DEO GLORIA

TABLE OF CONTENT

ACKNOWLEDGEMENTS i

TABLE OF CONTENT iii

SUMMARY xii

LIST OF TABLES xiv

LIST OF FIGURES xv

ABBREVIATIONS xx

CHAPTER ONE 1

INTRODUCTION 1

1.0 Objectives of the study 2

1.1 Gastrointestinal tract 6

1.1.1 Biology of intestinal mucosa 6

1.1.2 Mechanism of epithelium renewal 7

1.2 Intestinal immune system 9

1.2.1 Innate immunity 9

1.2.2 Structure of defensins 10

1.2.3 Mechanism of antimicrobial activity 11

1.2.4 Human alpha- and beta-defensins 11

1.3.1 Incidence, staging and survival rate of colorectal cancer 12

1.3.2 Classification of colorectal cancer 15

1.3.4 Treatment of colorectal cancer 17

1.3.4.1 Surgical resection 17

1.3.4.2 Chemotherapy 17

1.3.4.2.1 Mechanism of action of 5-FU 21

1.4 Tumor suppressors in colorectal cancer development 23

1.4.1 p53 23

1.4.2 DNA mismatch repair genes 25

1.5 Cell cycle checkpoints 27

1.5.1 The cell cycle complexes 27

1.5.2 CDK inhibitors 28

1.5.3 G1-S checkpoint 28

1.5.4 G2 checkpoint 29

1.6 Apoptosis 30

1.6.1 Caspase-dependent apoptosis 30

1.6.2 Clearance of apoptotic cells 31

1.7 Systemic Lupus Erythematosus 35

1.7.1 Role of autoantibodies 36

1.7.2 Impairment of apoptotic cell removal 37

CHAPTER TWO 39

MATERIALS AND METHODS 39

2.1 Cell lines and cell culture 40

2.1.1 Cell lines 40

2.1.1.1 PBMCs 40

2.1.1.2 Cell culture 41

2.1.2 Treatment of cells 41

2.1.2.1 Treatment of human colon cancer cells HT 29 with sodium butyrate 41

2.1.2.2 Treatment of human colon cancer cells HT 29 with glucose free medium41 2.1.2.3 Treatment of human colon cancer cells LS174T with dnTCF 42

2.1.2.4 Treatment of human colon cancer cells HCT 116 with genotoxic stressors

42

2.1.2.5 Treatment of HCT 116 cells with Nocodazole and Taxol 43

2.1.2.6 Treatment of HCT 116 cells with thymidine 43

2.1.2.7 Treatment of HCT 116 cells with caspase inhibitors 43

2.1.2.8 Treatment of human lymphocyte cell lines with UV 44

2.1.2.9 Treatment of PBMCs with gentoxic agents 44

2.1.2.10 Treatment of Jurkat cells with PHA/PMA 44

2.1.2.11 Treatment of U937 with PMA 45

2.1.3 Transient Transfection 45

2.1.3.1 Transient Transfection of plasmids 45

2.1.3.2 Transient transfection and dual-luciferase reporter assay 45

2.1.3.4 Transient transfection of siRNAs 46

2.2 Survival Assay 46

2.2.1 Colony formation assay 46

2.2.2 Propidium Iodide Staining 46

2.2.3 Annexin-V staining 47

2.2.4 Assay activities of caspase 3 47

2.3 RT-PCR 48

2.3.1 RNA isolation 48

2.3.2 cDNA synthesis 49

2.3.3 PCR reaction 49

2.3.4 Real-time PCR 49

2.3.5 Probe-based real-time RT-PCR 50

2.4 Cloning 51

2.4.1 DNA fragment purification by gel extraction 51

2.4.2 Restriction digestion 52

2.4.3 Ligation 52

2.4.4 Transformation 52

2.4.5 Plasmid miniprep 53

2.4.6 Plasmid midiprep 53

2.4.7 Sequencing reactions 54

2.4.8 Cloning of PRAP1 gene into mammalian and prokaryotic expression vectors 55

2.4.9 Cloning of PRAP1 promoter 55

2.4.10 Cloning of PRAP1 p53 binding elements 57

2.5 ELISA 58

2.5.1 Detection of anti-PRAP1 auto-antibody in serum 58

2.5.2 Detection of PRAP1 antigen in serum 58

2.5.3 Detection of cytokines 59

2.5.4 Cell-cell contact assay for cytokine production 59

2.6 Expression and purification of PRAP1 recombinant protein 60

2.6.1 Expression of GST-PRAP1 60

2.6.2 Purification of GST-PRAP1 protein 61

2.6.3 Expression and purification of His-tagged PRAP1 protein 61

2.7 Generation and purification of polyclonal antibody 62

2.8 Protein-protein interaction assay 64

2.8.1 Pulldown Assay 64

2.8.2 Immunoprecipitation 64

2.9 Immunodetection assay 65

2.9.1 Immunohistochemistry 65

2.9.2 Immunofluorescence microscopy 66

2.9.3 Scanning Electron microscopy 67

2.9.4 Western blotting 67

2.9.4.1 Protein extraction 67

2.9.4.2 Bio-Rad protein assay 68

2.9.4.3 SDS PAGE 69

2.9.4.4 Immunodetection 69

2.9.5 Immunodetection of anti-PRAP1 auto-antibody in serum 70

2.10 Bacteria binding assay 70

2.10.1 Detection by ELISA 70

2.10.2 Detection by direct binding 71

2.10.2.1 Labeling of protein with Alexa Fluor dye 71

2.10.2.2 Bacteria binding assay using labeled protein 71

2.11 DNA damage assay 72

2.11.1 Alkaline single-cell gel electophoresis (comet) assay 72

2.11.2 Micronucleus assay 73

2.12 Phagocytosis assay 73

2.12.1 Preparation of fluorescent beads 73

2.12.2 In vitro phagocytosis 73

2.13 Statistical Analysis 74

CHAPTER THREE 75

RESULTS 75

3.1 PRAP1 and intestinal differentiation 76

3.1.1 PRAP1 is expressed in epithelial cells of the intestines 76

3.1.2 Induction of PRAP1 by WNT-TCF pathway inhibition 79

3.1.3 Induction of PRAP1 by sodium butyrate 79

3.1.4 Induction of PRAP1 by glucose deprivation 82

3.2 Regulation of PRAP1 expression by differentiation 82

3.2.1 Induction of PRAP1 expression at mRNA level 82

3.2.2 Transcriptional regulation of PRAP1 85

3.2.2.1 Promoter characterization of PRAP1 85

3.2.2.2 PRAP1 expression was not regulated at transcriptional level 87

3.2.3 PRAP1 mRNA was stabilized in cellular differentiation 87

3.3 Effect of PRAP1 on differentiation 90

3.3.1 Effect of PRAP1 overexpression on cellular differentiation 90

3.3.2 Effect of PRAP1 knockdown on cellular differentiation 90

3.4 Role of PRAP1 in differentiated epithelial cells 93

3.4.1 PRAP1 binds bacteria 93

3.4.2 Bactericidal activity of PRAP1 95

3.4.3 Phagocytosis of bacteria 97

3.5 PRAP1 is a genotoxic stress responsive gene 97

3.5.1 Induction of PRAP1 by stressors that cause DNA damage 97

3.5.2 Transcriptional regulation of PRAP1 by genotoxic agents 99

3.5.3 Regulation of PRAP1 protein by genotoxic agents 102

3.5.4 Dose- and time-dependent regulation of PRAP1 105

3.6 Wild-type-p53-dependent induction of PRAP1 105

3.6.1 Genotoxic agents failed to induce PRAP1 in p53-/- cells 105

3.6.2 Restoration of PRAP1 induction by reintroduction of wild-type p53 in p53-/- cells 107

3.6.3 Genotoxic agents failed to induce PRAP1 in Hep 3B and HT 29 cells

107

3.7 PRAP1 is a novel p53-responsive gene 109

3.7.1 Identification of p53-response elements in PRAP1 gene 109

3.7.2 p53-response elements in PRAP1 gene are responsive to wild-type p53 113

3.8 PRAP1 modulates cell fate after genotoxic stress 113

3.8.1 Repression of PRAP1 induction by siRNAs 113

3.8.2 Effect of PRAP1 knockdown on colony formation 115

3.8.3 Effect of PRAP1 knockdown on sub-G1 118

3.8.4 Effect of PRAP1 knockdown on DNA damage 118

3.9 PRAP1 and cell cycle checkpoints 121

3.9.1 Enhanced cell death is accompanied by abrogation of S-phase arrest 121 3.9.2 Effect of PRAP1 knockdown on cyclins and CDKs 121

3.9.3 Effect of PRAP1 knockdown on cell cycle checkpoint proteins 124

3.9.4 Effect of PRAP1 knockdown on p53 level 127

3.9.5 PRAP1 expression was up-regulated in cells arrested at S-phase 127

3.9.6 PRAP1 inhibition in double-thymidine block assay 128

3.9.7 PRAP1 overexpression and cell cycle 129

3.9.8 PRAP1 knockdown in p53-/- cells 129

3.10 Mechanism of the cell death induced by PRAP1 inhibition 133

3.10.1 5-FU induced cell death is via a caspase-dependent mechanism 133

3.10.2 Inhibition of PRAP1 induces caspase-dependent apoptosis in cells treated with 5-FU 136

3.11 Other mechanisms employed by PRAP1 inhibition is enhancing cell death

138

3.11.1 Effect of PRAP1 on cytoskeleton 138

3.11.1.1 Effects of PRAP1 on cellular morphology 138

3.11.1.2 Effects PRAP1 on actin filament 139

3.11.1.3 Effects of PRAP1 on microtubules 141

3.11.2 PRAP1 interacts with Hsp 70 141

3.12 Role of PRAP1 in apoptotic cells 145

3.12.1 Induction of PRAP1 expression in apoptotic cells 145

3.12.2 PRAP1 binds to the surface of apoptotic cells 147

3.12.3 PRAP1 enhanced the phagocytosis of beads 153

3.13 Role of PRAP1 in a disease model, SLE 156

3.13.1 Detection of PRAP1 autoantigen 156

3.13.2 Detection of PRAP1 autoanitbodies 158

3.13.3 PRAP1 and proinflammatory cytokines 158

3.13.4 PRAP1 expression in PBMC 161

3.14 Regulation of PRAP1 expression in lymphocytes 165

3.14.1 PRAP1 is induced by PHA/PMA 165

3.14.2 PRAP1 is induced by UV 165

3.14.3 PRAP1 is induced by cytotoxic drugs 167

3.14.4 Regulation of PRAP1 expression in PBMC by cytotoxic drugs 167

CHAPTER FOUR 172

DISCUSSION 172

4.1 Role of PRAP1 in differentiated epithelial cells 173

4.1.1 Regulation of PRAP1 by differentiation 173

4.1.1.1 Expression of PRAP1 in intestinal epithelium 173

4.1.1.2 PRAP1 expression is positively correlated with differentiation 175

4.1.1.3 Regulation of PRAP1 expression by differentiation 177

4.1.2 Effect of PRAP1 on differentiation 178

4.1.3 PRAP1 and innate immunity 179

4.2 PRAP1, a p53-inducible modulator of cell fate in response to genotoxic

stress 182

4.2.1 PRAP1 is a genotoxic responsive gene 182

4.2.2 PRAP1 is a p53 responsive gene 184

4.2.3 PRAP1 modulates cell fate in response to genotoxic stress 186

4.2.4 Role of PRAP1 in cell cycle checkpoints 188

4.3 Role of PRAP1 in SLE 192

4.3.1 Induction of PRAP1 in apoptotic cells 192

4.3.2 Genotoxic agents failed to induce PRAP1 in PBMCs from SLE patients 193

CHAPTER FIVE 197

CONCLUSION 197

CONCLUSION 198

REFERENCES 200

APPENDIX I 213

APPENDIX II 218

SUMMARY

The Proline-rich acidic protein (PRAP1) was initially identified as a gene that was differentially expressed in pregnant mouse uterus The rat and human homologues were subsequently identified and found to be expressed abundantly in the gastrointestinal tract This thesis describes the characterization of the physiological role of human PRAP1 in the gastrointestinal tract and its possible role(s) in pathological states Broadly, PRAP1 has functions in two contrasting settings: life and death

The expression of PRAP1 is significantly correlated with the degree of differentiation of gastrointestinal epithelial cells In addition, PRAP1 is secreted into the gastrointestinal lumen and binds to the surface of bacteria It demonstrates bactericidal activity and may aid bacterial phagocytosis In colorectal cancer (CRC), PRAP1 is a downstream target of p53 and plays a protective role against genotoxic stressors Repression of PRAP1 significantly enhances the anti-tumor effect of chemotherapeutic drugs used in the treatment of CRC This is partly mediated through the abrogation of S-phase arrest in a p53 dependent manner, resulting in increased DNA damage and activation of the apoptotic pathway over cell cycle arrest Taken together, these imply that PRAP1 functions to maintain cell viability Conversely, there is also a possible role of PRAP1 during cell death

in facilitating the removal of apoptotic cells PRAP1 binds to the surface of apoptotic cells and may facilitate the clearance of apoptotic cells, the derangement

of which may lead to systemic lupus erythematosus (SLE)

In conclusion, PRAP1 is a multifunction protein with antimicrobial and cell fate modulation properties playing the contrasting roles of promoting cell

survival and cell death, which presents a novel target in the treatment of CRC and SLE.

LIST OF TABLES

Table 1.1 Definition of the T staging of the TNM classification 14

Table 1.2 Definition of the N and M staging of the TNM classification 15

Table 1.3 The five-year survival rate of 120,000 people diagnosed with colon cancer between 1991 and 2000 15

Table 1.4 Survival statistics for adenocarcinoma of the colon in Singapore 15

Table 1.5 Categories of drugs used in the management of colorectal cancer 19

Table 1.6 Commonly used bolus and infusional regimens used to administer 5-FU/LV chemotherapy 20

Table 1.7 Molecules implicated in apoptotic cell uptake 32

Table 2.1 Primer sequences for cloning of PRAP1 promoter 56

Table 2.2 Long template PCR system reaction mix 56

Table 2.3 Long Template PCR parameters 57

Table 2.4 Restriction digestion of vector 57

Table 2.5 Ligation reaction 57

Table 3.11 Sequences of the two p53 binding sites located in PRAP1 gene 111

LIST OF FIGURES

Figure 1.1 Sequences and the disulphide pairing of cysteines 10

Figure 1.2 Genetic model of colorectal carcinogenesis 16

Figure 1.3 Mechanism of action of 5-FU 23

Figure 1.4 DNA Damage Response 26

Figure 1.5 Regulation of cyclins 28

Figure 3.11 PRAP1 is expressed in the epithelial cells of small intestine 77

Figure 3.12 PRAP1 is expressed in the epithelial cells of colon 78

Figure 3.13 Differentiation is induced by blocking TCF4 80

Figure 3.14 PRAP1 is induced in differentiated colorectal cancer cells 80

Figure 3.15 PRAP1 expression is induced by sodium butyrate 81

Figure 3.16 PRAP1 expression is correlated with differentiation 81

Figure 3.17 PRAP1 expression is induced by glucose deprivation 83

Figure 3.18 PRAP1 expression is induced at mRNA level by differentiation 84

Figure 3.19 Cloning of PRAP1 promoter 84

Figure 3.20 PRAP1 promoter activities in L8 cells 86

Figure 3.21 PRAP1 is not regulated at transcriptional level by differentiation 88

Figure 3.22 Stability of PRAP1 mRNA is increased by differentiation 89

Figure 3.23 Overexpression of PRAP1 do not induce differentiation in HT 29 91

Figure 3.24 Overexpression of PRAP1 do not induce differentiation in L8 cells 92 Figure 3.25 Repression of PRAP1 expression did not affect differentiation in L8 cells 92

Figure 3.26 Purity of HisPRAP1 protein 94

Figure 3.27 HisPRAP1 binds to E.coli 94

Figure 3.28 HisPRAP1 binds to E.coli and Klebsiella 96

Figure 3.29 Bactericidial activity of HisPRAP1 at pH 4.5 96

Figure 3.30 Phagocytosis of E.coli 98

Figure 3.31 PRAP1 is induced by genotoxic stressors 100

Figure 3.32 Luciferase assay to identify the regions required for the prap1 gene promoter activity in HCT 116 cells 101

Figure 3.33 Identification of core promoter of prap1 gene 101

Figure 3.34 Induction of prap1 promoter activity by 5-FU and CPT 103

Figure 3.35 Induction of PRAP1 by 5-FU and CPT at mRNA level 104

Figure 3.36 PRAP1 was induced at protein level by 5-FU and CPT 104

Figure 3.37 Early upregulation of PRAP1 in a dose- and time-dependent manner 106

Figure 3.38 Early upregulation of PRAP1 protein by 5-FU and CPT 106

Figure 3.39 Induction of prap1 by 5-FU and CPT is dependent on p53 108

Figure 3.40 Reintroduction of wild-type p53 rescues the induction of prap1 by 5-FU and CPT in p53-/- cells 108

Figure 3.41 Hep 3B and HT 29 cells failed to induce prap1 gene expression 110

Figure 3.42 Schematic diagram of PRAP1 gene 111

Figure 3.43 Schematic diagram of p53 binding site of PRAP1 gene construct 112

Figure 3.44 Verification of the p53 binding sites constructed plasmid 112

Figure 3.45 Predicted p53 binding elements of PRAP1 response to wild-type p53 114

Figure 3.46 Suppression of PRAP1 induction by 5-FU at mRNA level 116

Figure 3.47 Suppression of PRAP1 induction by 5-FU at protein level 116

Figure 3.48 Repression of PRAP1 expression reduces colony numbers 117

Figure 3.49 Summary of the number of colonies formed 117

Figure 3.50 Repression of PRAP1 expression enhances the cell death induced by

5-FU 119

Figure 3.51 Repression of PRAP1 expression enhances 5-FU induced DNA damage measured by comet assay 120

Figure 3.52 Repression of PRAP1 expression enhances 5-FU induced DNA damage as measured by micronuclei assay 120

Figure 3.53 Repression of PRAP1 expression results in morphological changes 122

Figure 3.54 Repression of PRAP1 expression abrogates the S-phase arrest induced by 5-FU 123

Figure 3.55 Repression of PRAP1 expression reduces cyclin A1 125

Figure 3.56 PRAP1 knockdown reduces CDK2 125

Figure 3.57 Repression of PRAP1 expression affects p21 localization 126

Figure 3.58 PRAP1 was upregulated in S-phase arrested cells 128

Figure 3.59 Abrogation of the S-phase arrest in double-thymidine block 130

Figure 3.60 Overexpression of PRAP1 alone failed to induce S-phase arrest 131

Figure 3.61 Repression of PRAP1 in p53-/- cells failed to abrogate S-phase arrest 132

Figure 3.62 Repression of PRAP1 in p53-/- cells failed to enhance cytotoxicity of low dose of 5-FU 132

Figure 3.63 Treatment of p53-/- cells with high dose of 5-FU 134

Figure 3.64 5-FU induces caspase-dependent apoptosis in HCT 116 135

Figure 3.65 Repression of PRAP1 induces caspase 3 activity in 5-FU treated cells 135

Figure 3.66 Repression of PRAP1 enhances caspase-dependent apoptosis 137

Figure 3.67 Effects of PRAP1 on actin network 140

Figure 3.68 Effects of PRAP1 on microtubulin network 142

Figure 3.69 GST-PRAP1 pull-down 143

Figure 3.70 Identification of PRAP1 binding protein by MS-MS 144

Figure 3.71 PRAP1 physically interacts with Hsp 70 144

Figure 3.72 Dying cells in floating population 146

Figure 3.73 Apoptotic cells in floating population 148

Figure 3.74 PRAP1 expression is increased in apoptotic cells 148

Figure 3.75 Immunofluorescence images showing the binding of PRAP1 to the surface of apoptotic cells 150

Figure 3.76 Histogram showing PRAP1 binds to the surface of apoptotic cells 150 Figure 3.77 PRAP1 binds on the surface of HCT 116 apoptotic cells 151

Figure 3.78 Alexa fluor labeled HisPRAP1 binds directly to the surface of apoptotic cells 151

Figure 3.79 Transmission electron microscopy of PRAP1 on apoptotic cells 152

Figure 3.80 Flow cytometry analysis of phagocytosis of beads by U937 154

Figure 3.81 HisPRAP1 enhances phagocytosis of beads by macrophages 155

Figure 3.82 PRAP1 protein was detected in the serum of both normal and SLE patients 157

Figure 3.83 PRAP1 autoantibody was detected in serum 159

Figure 3.84 Verification of PRAP1 autoantibodies in the serum 160

Figure 3.85 Effects of low concentration of HisPRAP1 protein on the production of proinflammatory cytokines 162

Figure 3.86 Effects of high concentration of PRAP1 on the production of proinflammatory cytokines 163

Figure 3.87 PRAP1 mRNA expression is reduced in SLE patients 164

Figure 3.88 PRAP1 expression is induced by PHA/PMA 166

Figure 3.89 PRAP1 expression is induced by UV 166

Figure 3.90 PRAP1 expression is induced by cytotoxic drugs in lymphocytes 168Figure 3.91 PRAP1 is induced in normal PBMC by cytotoxic drugs 169Figure 3.92 PBMCs from SLE patients failed to induce PRAP1 169Figure 3.93 PBMCs from SLE patients failed to induce PRAP1 mRNA 170

3-[(3-cholamidopropyl1)dimethyammonio]-1-CLL chronic lymphocytic leukemia

CRC colorectal cancer

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dnTCF dominant negative TCF

dNTP deoxyribonucleotide triphosphate

dsDNA double stranded DNA

DTT dithioreitol

EDTA ethylenediaminetetraacetic acid

ELISA enzyme linked immunosorbent assay

ER endoplasmic reticulum

FAP familial adenomatous polyposis

FBS fetal bovine serum

FITC fluorescein isothiocyanate

GST glutathione S-transferase

HDAC histone deacetylase

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HSP heat shock protein

Nab natural antibody

NaCl sodium cholride

PBMC peripheral blood mononuclear cell

PBS phospate buffered saline

PMSF phenylmethylsulfonyl fluoride

RNA ribonucleic acid

RT-PCR reverse transcriptase polymerase chain reaction

SD standard deviation

SDS sodium deodecyl sulphate

SDS-PAGE SDS-polyacrylamide gel electrophoresis

SEM standard error of the mean

siRNA small interference RNA

SLE systemic lupus erythematosus

TBE tris-borate EDTA

TCF T-cell factor

TNF tumor necrosis factor

UTR untranslated region

CHAPTER ONE

INTRODUCTION

1.0 Objectives of the study

Proline rich acidic protein gene (PRAP1) was first identified in the mouse

(Kasik and Rice 1997) The gene was expressed in the mouse uterus from day 12

of gestation to the third day after parturition Later, our group investigated the rat homologue of PRAP1, and showed that the expression of PRAP1 in rat was not limited to late pregnant uterus There was abundant expression in the mucosa of gastrointestinal tracts of mouse and rat (Zhang, Rajkumar et al 2000) The human homologue of PRAP1 was subsequently isolated and characterized (Zhang, Wong

et al 2003) by our group Human PRAP1 was also found to be abundantly expressed in the epithelial cells of the gastrointestinal tract and cervix In contrast

to mouse and rat, human PRAP1 was not restricted to the gastrointestinal tract, butwas also found in the hepatocytes of the liver, and proximal and distal renal tubules The significance of the species-specific localization of PRAP1 remains unknown, but it may suggest that PRAP1 have different functions in different species and organ systems

Both human and rodents have PRAP1 expression in epithelial cells of the gastrointestinal tract, suggesting that PRAP1 may be associated with epithelial cell differentiation PRAP1 was also used as a differentiation marker by other studies (Zhang, Wong et al 2003; Tou, Liu et al 2004) However, its functional role in the differentiation process has not been elucidated Hence, the first main objective of this study is to further validate the association of PRAP1 with intestinal differentiation and its possible role in the process of differentiation To achieve this, the following issues were examined:

1 expression pattern and localization of PRAP1 in normal human small intestine and colon using immunohistochemical staining

2 correlation of PRAP1 expression with three well-characterized differentiation models, namely WNT/TCF pathway, glucose deprivation and sodium butyrate

3 effect of perturbing PRAP1 expression on the differentiation process

Beyond expression in physiological states, our group has previously found that PRAP1 expression was regulated epigenetically and was down-regulated in right-sided colon adenocarcinoma when compared with the respective adjacent normal tissues Furthermore, overexpression of PRAP1 was reported to inhibit the growth of colon cancer cell lines (Zhang, Wong et al 2003), suggesting that PRAP1 may play a negative regulatory role in the development of colorectal cancer

Colorectal cancer is the world's third commonest cancer, and second most common cancer in Singapore There is an increasing understanding of the molecular pathology of this cancer of the large bowel Primarily, the diagnosis of colon cancer is made through colonoscopy with biopsy The mainstay of treatment

is removal of the tumor by surgery, followed by adjuvant chemotherapy

The chemotherapeutic agent, 5-Fluorouracil (5-FU) has been used as the first-line therapy for advanced colorectal cancer and as adjuvant therapy for early colorectal cancer ever since it was first discovered 40 years ago (Poon, O'Connell

et al 1989; Giacchetti, Perpoint et al 2000; Gill, Loprinzi et al 2004) However,

in patients with advanced colorectal cancer, only a subset of tumors responded to 5-FU (10 to 15%, Johnston and Kaye 2001) Although its enzymatic conversion and metabolism are well defined, the cellular factors that determine the sensitivity

or resistance to this drug remain poorly understood Hence, finding new genes and

new functions of known genes that modulate the phramacokinetics and pharmacodynamics of chemotherapeutic agents will no doubt help shed new light

in the treatment of colorectal cancer Thus, the second main objective of this thesis is to investigate whether PRAP1 plays a role in modulating the efficacy of these chemotherapeutic drugs especially 5-FU To address this, the following issues were examined:

1 regulation of PRAP1 gene by various stressors at the mRNA and protein levels

2 regulation of PRAP1 by p53

3 PRAP1 in modulating the efficacy of 5-FU

4 PRAP1 and cell cycle arrest

5 PRAP1 and cytoskeleton

6 PRAP1 binding protein

In addition to these two main objectives, our work has identified two novel functions of PRAP1, which were further explored in this thesis First is its role as

an antimicrobial protein The following issues were addressed:

1 the binding of PRAP1 to the surface of bacteria

2 the bactericidal activity of PRAP1

3 the effect of PRAP1 on the phagocytosis of bacteria

The second novel function of PRAP1 studied is its role in the clearance of apoptotic cells, with an aim to address the following issues:

1 the expression of PRAP1 in apoptotic cells

2 the binding of PRAP1 to the surface of apoptotic cells

3 the effect of PRAP1 on the phagocytosis of beads

4 its reverence in the disease setting of systemic lupus erythematosus (SLE)

i the detection of PRAP1 antigen and autoantibodies in the serum of SLE patients

ii the effect of PRAP1 on the production of proinflammatory cytokines

iii the expression of PRAP1 in peripheral blood mononuclear cells (PBMCs) of SLE patients

iv the regulation of PRAP1 expression in apoptotic cells of lymphocyte cell lines, PBMCs of normal and SLE patients

1.1 Gastrointestinal tract

1.1.1 Biology of intestinal mucosa

The gastrointestinal tract has a well-defined architecture It is made up of a muscular tube lined by a mucous membrane with a relatively constant arrangement of the major components throughout the tract The intestinal tract is made up of four distinct functional layers: mucosa, submucosa, muscularis propria and adventitia The intestinal mucosa has three components: the epithelium, the supporting lamina propria and a thin smooth muscle layer, the muscularis mucosae The small intestine is typically made up of absorptive mucosa which is arranged into finger-like projections called villi, with intervening short glands called crypts The intestinal villi are lined by a simple columnar epithelium that continues with that of the crypts; whereas the mucosa of the colon is arranged into closely packed and straight tubular glands containing cells specialized for water absorption and mucus secretion

The intestinal epithelium has a variety of cell types, each with its own specific function The main cell types include: the predominant enterocytes that are tall columnar cells with surface microvilli which are seen as brush border in the light microscope; the mucus secreting Goblet cells that are scattered among the enterocytes; hormones secreting neuroendocrine cells; Paneth cells usually found in the small intestine which play a vital role in innate immunity; stem cells that are located at the base of the crypts which proliferate vigorously to replenish all the above cell types and intraepithelial lymphocytes which are mostly T cells that provide defense against invasive organisms

The epithelium of gastrointestinal tract is a dynamic tissue which renews itself rapidly At the surface of intestinal epithelium, cells undergo apoptosis and/or extrusion into the lumen Stem cells at the crypt bottom replenish the dead cells through a coordinated series of events involving proliferation, lineage differentiation and migration to the villus or intercrypt table This whole process

takes place every 3 to 5 days (Potten and Loeffler 1990)

1.1.2 Mechanism of epithelium renewal

Studies by the laboratory of Clevers have shed light on the mechanisms that control cell fate determination of intestinal epithelial cells and their directional migration and specific positioning (van de Wetering, Sancho et al 2002)

WNT signaling pathways play a central role in controlling the switch between proliferation and differentiation (Pinto and Clevers 2005) in the intestinal epithelium A large number of proteins are involved in the regulation of the WNT signaling and consequent cellular responses The WNT signaling pathway begins with the binding of WNT proteins to cell surface receptors of the Frizzled family This interaction results in the activation of the Dishevelled family proteins (DSH) which are key components of a membrane-associated WNT receptor complex Activated DSH inhibits a second protein complex consisting of axin, glycogen synthase kinase 3 (GSK-3) and adenomatosis polyposis coli (APC) The axin/GSK-3/APC complex tightly regulates the level of β-catenin by phosphorylating β-catenin and promoting its ubiquitination and subsequent degradation by proteasomes In the presence of WNT signaling, this β-catenin destruction complex is inhibited, leading to the accumulation and stabilization of β-catenin β-catenin enters the nucleus and interacts with TCF/LEF family of

transcription factors to drive specific gene expression Genes activated by WNTs are expressed in the proliferative compartment of crypts and are overexpressed in colorectal tumors Genes that are repressed by WNTs were expressed in specialized cells at the top of crypts and in villi

In cancers, mutations in the β-catenin destructive complex have been reported Truncating mutations in APC and axin, and mutations in GSK-3, all lead

to the formation of constitutive nuclear β-catenin/TCF complexes (Korinek, Barker et al 1997; Morin, Sparks et al 1997; Rubinfeld, Robbins et al 1997; Satoh, Daigo et al 2000) Mutational activation of β-catenin/TCF in intestinal epithelial cells leads to the formation of polyps, the first morphological alteration that ultimately results in colorectal cancer (Kinzler and Vogelstein 1996) Tcf-4 is the most predominantly expressed TCF family member in the intestinal epithelium and is required to establish the proliferative progenitors in the fetal small intestinal epithelium (Korinek, Barker et al 1998)

In summary, β-catenin/TCF signaling is essential for maintaining the proliferative/undifferentiated state of the intestinal epithelial cells, and disruption

of this contributes to the early stage of intestinal tumorigenesis

In addition to its role in controlling the switch between proliferation and differentiation, β-catenin/TCF signaling also mediates cell positioning in the intestinal epithelium by controlling the expression of EphB (receptor) and Ephrin

B (ligand) The expression of EphB2 and EphB3 are restricted to the cells in the intervillus regions while Ephrin B1 is expressed in a complementary pattern by the adjacent cells in the villus Both receptors and ligand are coexpressed in the proliferative cells bordering the intervillus pockets The interaction of EphB and ephrin ligands frequently result in cell repulsion, such that cells with the ligands

expressed on their surface are sorted away from cells expressing the receptor Clevers group demonstrated that in mice with mutations in EphB2 and EphB3, the different cell types in the intestinal epithelium are mispositioned (Batlle, Henderson et al 2002) The proliferative cells are no longer restricted to the crypts and Paneth cells do not follow their downward migratory path, but scatter along the crypt and villus

In conclusion, the WNT/TCF signaling pathway is a central mechanism that controls cell fate determination and positioning in the intestinal epithelium

1.2 Intestinal immune system

1.2.1 Innate immunity

A large number of indigenous microbial flora resides in the human intestinal tract, with majority of them present in the colon Besides these indigenous microbes, the gastrointestinal tract is constantly exposed to a variety of bacteria, including some potential pathogens, from the ingestion of food and drink Innate immune mechanisms are thought to be involved in protecting the host from invasion by luminal bacteria Paneth cells at the base of crypts are recognized as effectors of the intestinal innate immunity These cells secrete a variety of antimicrobial peptides and proteins, which play an important role in intestinal mucosal innate immunity A number of these peptides and proteins have been identified such as enteric α-defensins (Ouellette and Selsted 1996), lysozyme (Mason and Taylor 1975) and secretory phospholipase A2 (Harwig, Tan et al 1995)

1.2.2 Structure of defensins

Defensins are small (15-20 residues, 2-6 kDa) cysteine-rich cationic proteins containing three pairs of intramolecular disulfide bonds They are active against bacteria, fungi and enveloped viruses Mammalian defensins are classified into alpha, beta and theta based on their size and pattern of disulfide bonding (Figure 1.1) The six cysteines in α-defensins are linked in the 1-6, 2-4 and 3-5 pattern, whereas in β-defensins the pattern is 1-5, 2-4 and 3-6 Their structures have been solved by two-dimensional NMR and by X-ray crystallography Both alpha and beta defensins consist of a triple-stranded β-sheet with a distinctive fold, whereas the structure of theta defensins is cyclic, forming a simple β-sheet (Ganz 2003)

C

R

C

R T G F

C

R θ-defensin

C

R

C

R T G F

C

R

C R C L C

R R G V

C

R

C

R T G F

C

R θ-defensin

Figure 1.1 Sequences and the disulphide pairing of cysteines

Solid line indicates the peptide bond between the two hemi-defensins Adapted from Nature Reviews Immunology 3, 710-712, 2003

1.2.3 Mechanism of antimicrobial activity

Defensins work by permeabilizing target membranes A model called the carpet-wormhole is proposed from experiments using artificial membranes In this model, defensins that are amphipathic have clusters of positively charged amino acid side chains and hydrophobic amino acid side chains This allows them to interact with microbial membrane with their negatively charged phospholipid headgroup and hydrophobic fatty acid chains The electrostatic attraction and the transmembrane bioelectric field pull the defensin peptides towards and into the membrane As the peptides accumulate in a ‘carpet’, the membrane is strained and the peptides transited into another arrangement that lowers the strain but results in the formation of membrane ‘wormholes’ or pores (Ganz 2003) However, this model is complicated by the marked differences in net charge, amino-acid sequence and quaternary structure among defensins These differences may have evolved so that various defensins can target different types of bacteria with differing structures of cell walls and membranes

1.2.4 Human alpha- and beta-defensins

Enteric α-defensins are also termed as cryptdins with more than 17 isoforms found in murine Paneth cells, and two in human, human defensin 5 and 6 (HD-5 and HD-6) HD-5 is expressed in Paneth cells of the normal duodenum, jejunum and ileum, but not found in normal stomach and colon, suggesting an important role in protecting the small intestine against bacteria HD-5 is not only expressed in Paneth cells, but also in the rare epithelial cell type called the intermediate cell HD-5 is stored as a precursor form in the granules of Paneth cells, and cleaved by trypsin and processed to its mature form HD-6 has similar

distribution and processing as HD-5 and even has similar ionic charge properties

to HD-5, but unlike HD-5 it has been showed to have very poor antibacterial activity in vitro (Ericksen, Wu et al 2005)

In addition to cryptdins, neutrophil α-defensins, HNP 1-3, are found to be expressed in intestinal epithelial cells (Cunliffe, Kamal et al 2002) Both cryptdins and neutrophil α-defensins are active against a broad spectrum of bacteria and fungi (Ganz, Selsted et al 1985; Ouellette and Selsted 1996)

In contrast to the restricted expression of α-defensins, human β-defensins are expressed at multiple sites including intestinal epithelial cells The expression of β-defensins is inducible at sites of infection or inflammation The most abundant β-defensins, hBD-2 and hBD-3 are expressed mainly in stomach and colon and have bactericidal activity against a variety of bacteria Besides their bactericidal activity, human β-defensin 1 and 2 (hBD-1 and hBD-2) have chemoattractant activity for cells expressing the chemokine receptor CCR-6, such as dendritic cells (Yang, Chertov et al 1999) This serves as a bridge between the innate immunity

at the intestinal mucosa and subsequent adaptive immune response Dendritic cells are able to internalize foreign materials and to present antigens to naive T and B lymphocytes, either locally or systemically through the lymphatic drainage system The presence of other epithelial antimicrobial peptides has been described This suggests a degree of redundancy within the innate defense system

of the intestinal tract

1.3 Colorectal Cancer

1.3.1 Incidence, staging and survival rate of colorectal cancer

Colorectal cancer (CRC) is the third commonest cancer in the world, and second most common cancer in Singapore According to the latest figures released

by the Singapore Cancer Registry (2004), about 500 Singaporeans develop colon cancer and 300 Singaporeans develop rectal cancer yearly The numbers are increasing every year and mirrored those observed in developed countries such as

The T categories of CRC describe the extent of spread through the layers that form the wall of the colon and rectum These layers include the inner lining (mucosa), the thin muscle layer (muscularis mucosa), the fibrous tissue beneath the muscle layer (submucosa), the thick muscle layer (muscularis propria) and the thin, outermost layers of connective tissue (subserosa and serosa) The T staging

of the TNM system is defined in Table 1.1 The definition of the different staging

of N and M categories are also included in Table 1.2 Table 1.3 shows the year survival rate of patients belonging to the different stage groupings All the information pertaining to AJCC/TNM staging was obtained from the American

Cancer Society website accessed on August 2008 Table 1.4 indicates the survival rate for CRC patients in Singapore

five-The survival rate for patients with colon cancer is higher when the disease

is detected at an early stage At Stage 0 and 1, majority of patients have a survival rate of close to 100% (Table 1.3) Hence, studies focusing on the enhancement of early detection of this disease are absolutely necessary to improve the overall survival rate of the patients

The worst prognosis for colon cancer is when patients are diagnosed with metastasis The five-year survival rate dropped drastically to 8-12% (Table 1.3 and 1.4) Metastasis is a complex, multigenic process The elucidation of molecular mechanisms leading to metastasis will provide greater insights into the understanding of the physiological pathways deranged in metastasis In addition, identification of new chemotherapeutic drugs, targeted therapies, and optimizing the current regimens are also vital in the treatment of patients with advanced colon

cancer

Category Definition

Tx No description of the tumor's extent is possible because of incomplete

information

Tis The cancer is in the earliest stage It involves only the mucosa It has

not grown beyond the muscularis mucosa (inner muscle layer)

T1 The cancer has grown through the muscularis mucosa and extends

into the submucosa

T2 The cancer has grown through the submucosa and extends into the

muscularis propria (outer muscle layer)

T3 The cancer has grown through the muscularis propria and into the

subserosa but not to any neighboring organs or tissues

T4 The cancer has grown through the wall of the colon or rectum and into

nearby tissues or organs

Table 1.1 Definition of the T staging of the TNM classification

Categories Definition

Nx No description of lymph node involvement is possible because of

incomplete information

N0 No lymph node involvement is found

N1 Cancer cells found in 1 to 3 nearby lymph nodes

N2 Cancer cells found in 4 or more nearby lymph nodes

Mx No description of distant spread is possible because of incomplete

information

M0 No distant spread is seen

M1 Distant spread is present

Table 1.2 Definition of the N and M staging of the TNM classification

Stage Grouping TNM designation Five-year survival rate

Table 1.3 The five-year survival rate of 120,000 people diagnosed with colon cancer between 1991 and 2000 Date obtained from a study of the National

Cancer Institute’s SEER database

Staging First five-year survival rate

(Male) (Female)

Table 1.4 Survival statistics for adenocarcinoma of the colon in Singapore

Data obtained from patients registered in the Singapore Registry from 1968 to

1997

1.3.2 Classification of colorectal cancer

There is an increasing understanding of the molecular pathology of the cancer of the large bowel CRC can be classified into sporadic or familiar

Familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC) are the two best characterized familial CRC

About 90-95% of them is sporadic (de la Chapelle and Peltomaki 1995) and thought to develop according to the Vogelstein model of carcinogenesis (Figure 1.2) In this model, Vogelstein described the sequential events of gene defects that lead to the transition of normal mucosa to adenoma to carcinoma and finally metastasis The genes involved include adenomatous polyposis coli (APC), K-ras and p53 (Fearon and Vogelstein 1990) There are also reports on alternative mechanisms of carcinogenesis such as abnormal DNA methylation and histone deacetylation

Intermediate adenoma

Late adenoma

β-catenin axin

Genomic instability

Intermediate adenoma

Late adenoma

Carcinoma Metastasis Normal

epithelium

Aberrant

Crypts foci

Early adenoma

Intermediate adenoma

Late adenoma

β-catenin axin

Genomic instability

Figure 1.2 Genetic model of colorectal carcinogenesis

Adapted from Cell 61: 759-767, 1990

The other 5 to 10% of bowel carcinomas are hereditary, including familial adenomatous polyposis and hereditary non-polyposis colorectal cancer FAP is an autosomal, dominantly inherited disease About 95% of the gene carriers have been shown to have mutations in the APC gene (Groden, Thliveris et al 1991) HNPCC is caused by inherited mutations in DNA mismatch repair genes such as mutL homolog 1 (Hmlh1), which will lead to microsatellite instability (Houlston and Tomlinson 1997) This microsatellite instability is also observed in about 15%

of sporadic colorectal cancers Sporadic right sided colorectal cancer is found to

have more microsatellite instability than left sided bowel cancer On the other hand, left sided colorectal cancer is observed to show more features of loss of heterozygosity, mutation in p53, up-regulation of vascular endothelial growth factor (VEGF) and aneuploidy

The genetic variability in these tumors is relevant in the selection of treatment schemes Left-sided cancers that have mutated p53 and over-expressed VEGF are reported to be associated with a poor prognosis and poor response to fluorouracil based chemotherapy Newer agents such as irinotecan (camptothecin, CPT) and antibody against VEGF may be appropriate for these tumors For right-sided cancers with wild-type p53, 5-flurouracil (5-FU) would be a good choice (Richman and Adlard 2002)

1.3.4 Treatment of colorectal cancer

1.3.4.1 Surgical resection

Treatment of colorectal cancer is dependent on its stage at the time of diagnosis The primary treatment is surgical removal of polyps or tumors with sufficient margins, and radical en-bloc resection of mesentery and lymph nodes to reduce local recurrence In patients with multiple metastases, palliative resection

of primary tumor is recommended to reduce further morbidity resulting from tumor bleeding, invasion and its catabolic effect The removal of isolated liver

metastases by surgery is often offered

1.3.4.2 Chemotherapy

Chemotherapy and/or radiotherapy may be used together depending on the individual colorectal cancer patent’s staging and other medical factors Most chemotherapeutic drugs target the ability of cell to grow or to multiply Therefore,

tumors with high growth fractions are more sensitive to chemotherapy, whereas malignancies with slower growth rates have poor response However, most chemotherapeutic drugs also affect normal rapidly dividing cells such as cells for hair growth and for replacement of intestinal epithelium, causing side-effects in patients such as hair loss, vomiting, and diarrhea

Chemotherapy is usually given with a curative intent or to palliate symptoms or aim to prolong life When it is given as a neoadjuvant (chemotherapy prior to surgery), it is aimed at shrinking the primary tumor, thereby rendering local therapy (surgery or radiotherapy) less destructive or more effective It is given as an adjuvant (chemotherapy after surgery) to reduce chances of recurrent after the removal of tumor by surgery or radiotherapy or to kill any cancerous cells that have spread to other parts of the body As a palliative therapy, it is used simply to reduce tumor size and prolong life (Skeel 2003) In CRC, if the cancer has spread to the lymph nodes (Stage III), adjuvant chemotherapy is usually given

More than 50 chemotherapeutic drugs are currently available to treat cancer and they are classified based on their mechanisms of action The main types of chemotherapy drugs are: alkylating agents such as cisplatin that directly attack DNA; antimetabolites such as 5-FU that interfere with the production of DNA and keep cells from growing and multiplying; antitumor antibiotics such as doxorubicin that interfere with important cell functions, including the production

of DNA and cell proteins; plant alkaloids such as vinblastine and taxol that block cell division by preventing microtubule function; topoisomerase inhibitors such as camptothecin and etoposide that interfere with both transcription and replication

of DNA by disrupting proper DNA supercoiling; other agents that do not interfere