i ACKNOWLEDGEMENTS I would like to express my gratitude to the following people for their contribution and for making this project possible: My main supervisor, Associate Professor Hooi Shing Chuan, for his valuable guidance and persistent support, without which, this thesis would not have been produced in the present form. I am grateful for the many opportunities given to me in exploring the area of research in molecular biology. I have learnt much in the past six years that I have spent in this laboratory. 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. ii 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 iii TABLE OF CONTENT ACKNOWLEDGEMENTS i TABLE OF CONTENT . iii SUMMARY .xii LIST OF TABLES xiv LIST OF FIGURES . xv ABBREVIATIONS . xx CHAPTER ONE INTRODUCTION . 1.0 Objectives of the study . 1.1 Gastrointestinal tract 1.1.1 Biology of intestinal mucosa 1.1.2 Mechanism of epithelium renewal 1.2 Intestinal immune system 1.2.1 Innate immunity 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 iv 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 v 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 . 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 vi 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 vii 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 viii 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 ix 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 x 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 59 incubated with 50µL of donkey anti-rabbit IgG conjugated-HRP (1:500 in assay diluent) for hour at room temperature. The plate was washed times with PBST and 100µl of TMB substrate buffer was added and incubated for 30 minutes and 100µL of 1M H2SO4 was added to stop the reaction. Absorbance was read at 450nm with reference wavelength at 570nm. 2.5.3 Detection of cytokines Human IL-6, IL-1β and TNFα (BD Biosciences) were assayed by ELISA using paired antibodies according to the manufacturer’s instructions. Microwells were coated with 100µL of Capture antibody diluted in coating buffer and incubated overnight at 4oC. The wells were washed three times with PBST and blocked with 200µL of assay diluent for hour at room temperature. One hundred microliters of standards and samples diluted in assay diluent were added and incubated for hours at room temperature. The plate was washed five times with PBST. 100µL of biotinylated Detector antibody diluted in assay diluent was added and incubated for hour. The plate was washed seven times with PBST. One hundred microliters of Avidin-HRP diluted in assay diluent was added and incubated for 30 minutes. The plate was washed with PBST for seven times. 100µL of TMB substrate buffer was added and incubated for 30 minutes at room temperature in the dark. One hundred microliters of 1M H2SO4 was added to stop the reaction. Absorbance was read at 450nm with reference wavelength at 570nm. 2.5.4 Cell-cell contact assay for cytokine production Jurkat cells were seeded at one million per mL density in a T75 flask and treated with PHA and PMA for 48 hours. Cells were harvested by spinning at 180xg for minutes at 4oC and washed twice with PBS/EDTA. Cells were next 60 fixed with 1% paraformaldehyde on ice for to hours. Cells were washed twice subsequently with PBS/EDTA. The fixed cells were diluted to a density of × 106 cells/mL in complete media and seeded 50µL per well in a 96-well plate. U937 cells were harvested and diluted to a density of × 105 cells/mL in complete media and seeded 100µL per well into wells with fixed Jurkat cells. Next, 50µL of His-PRAP1 proteins that were diluted in various concentrations in complete medium were added into each well to a final volume of 200µL. Cells were then cultured for 48 hours. Cell culture supernatant was harvested and stored at -20oC before the measurement of cytokine production was taken by ELISA assay. 2.6 Expression and purification of PRAP1 recombinant protein 2.6.1 Expression of GST-PRAP1 The pGEX-5X PRAP1 plasmid was transformed into BL21 competent cells and plated unto LB plates and incubated at 37oC overnight. In order to optimize the expression, one clone was picked and was grown in separate tubes and induced with different concentration of IPTG (Invitrogen). Pellets of the cultures were lysed and the lysate was run on SDS-PAGE gel to identify the concentration with the highest expression level. Next, 10 colonies were picked and induced with the optimized concentration of IPTG (0.1mM) and an aliquot of the cultures were lysed and checked on SDS-PAGE gel for the identification of the clone with the highest expression level. The clone with the highest expression of GST-PRAP1 was inoculated and grown in 10mL LB medium for 12-15 hours at 37oC with vigorous shaking. Next, the culture was diluted 1:100 into fresh pre-warmed LB medium and grown at 37oC with shaking until the reading of OD600 at 0.5-0.6 was achieved. 61 IPTG was then added to a final concentration of 0.1mM and was left to culture for another hours. The culture was then centrifuged at 10,000xg for 10 minutes using a Beckman JA 14 rotor at 4oC. The bacteria pellet was resuspended in 50mL ice-cold PBS containing 1mM PMSF and 0.5mM EDTA. The suspended cells were lysed by sonication for 10 minutes on ice in short bursts. 2.5mL of Triton X100 (20%) was added to aid in solubilization of the fusion protein for 30 minutes. The lysate was then centrifuged at 12,000xg for 10 minutes at 4oC. The supernatant was transferred to a fresh tube for purification. 2.6.2 Purification of GST-PRAP1 protein Five milliliters of 50% slurry of Glutathione Sepharose 4B (Amersham) was prepared in PBS and added to the cleared lysate. The mixture was incubated for 30 minutes at room temperature with end to end rotation and was transferred to a disposable polypropylene column (Qiagen). The unbound lysate was drained by gravity flow. The column was washed twice with 10 bed volumes of PBS. The fusion protein was eluted by adding 5mL of elution buffer containing 10mM reduced glutathione in 50mM Tris-HCl. All eluted fractions were analyzed on SDS-PAGE gel. The fractions with high purity and concentration were pooled together and dialyzed against 3L PBS. The dialysis buffer was changed every hours. The protein concentration was measured by Bio-Rad protein assay and purity was checked by running on SDS-PAGE gel and visualized by Coomassie Blue staining solution (0.05% Coomassie R250, 40% ethanol, 10% acetic acid). 2.6.3 Expression and purification of His-tagged PRAP1 protein The pET-19b PRAP1 was transformed into BL21 competent cells. Similar to the optimization procedures of GST-PRAP1 expression, IPTG concentration 62 was optimized and one clone with the highest expression was identified. The large culture growth and centrifugation were done in the same way as that of GSTPRAP1. For the lysis of bacteria pellet, 50mL of lysis buffer (50mM Na2HPO4, 300mM NaCl, 10mM Imidazole, pH 8) containing 1mM PMSF was added and incubated on ice for 30 minutes. The solution was sonicated on ice for minutes with 10 seconds cooling period between each 10 seconds burst. The lysate was centrifuged at 10,000xg for 20 minutes. FPLC was performed according to the manufacturer's instructions (AKTA; Amersham). The lysate was loaded into a sample super loop. The lysate was subsequently loaded into the 5mL NiNTA column (Amersham) at a rate of 0.5mL/minutes. The column was washed with buffer A (50mM Na2HPO4, 300mM NaCl) at increasing strength of buffer B (250mM Imidazole). The washing procedure was as follows: CV (column volume) of 4% B, CV of 20% B and finally CV of 50% B. The His-PRAP1 protein was eluted with CV of 100% B. The peak fractions were collected and analyzed on SDS-PAGE gel. Fractions with high purity and concentration were pooled together and dialyzed against PBS. 2.7 Generation and purification of polyclonal antibody Two female New Zealand white rabbits were used for immunization. 500µg of GST-PRAP1 protein was emulsified with complete Freund’s adjuvant (1:1). Rabbits were anesthetized and antigen was injected subcutaneously into sites. The rabbits were immunized every fortnightly. From second injection onwards, the antigen was emulsified in incomplete Freund’s adjuvant. After immunizations, rabbits were bled from the ear vein to collect serum. The serum was affinity purified with His-PRAP1 protein immobilized onto CNBr column. 0.1g of dried CNBr-activated sepharose Fast Flow beads 63 (Amersham Biosciences) was swelled in 10mL of 1mM HCl for 30 minutes. The beads were washed two times with 1mM HCl for minutes each, followed by washes in coupling buffer (0.2M NaHCO3, 0.5 M NaCl pH 8.3). His-PRAP1 protein was diluted in coupling buffer and immobilize unto the beads in a mL tube. The reaction mix was incubated for hours with mixing. The unbounded protein was washed off with gel volumes of coupling buffer for times. The excess activated sites were blocked with 5mL of 1M Ethanolamine pH at 4oC overnight. The beads were then packed into a 1mL disposable polypropylene column (Qiagen). The column was washed two times in Buffer A, followed by two times in buffer B (A: 0.1M sodium acetate 0.5M NaCl pH 4, B: 0.1M Tris 0.5M NaCl pH 8). Then the column was washed two times with PBS. The rabbit serum was diluted in PBS (1:1) and flow through the column by gravity at 4oC. The unbounded antibodies were washed with column volumes of PBS for times. Antibody was eluted using 0.1M Na Citric acid pH 3.04 and neutralized with 10M NaOH and stored at -20oC with 0.01% sodium azide. Alternatively, antibodies were purified using Gentle Ag/Ab binding and Elution Buffers (Pierce) according to the manufacturer’s instructions. The CNBr-PRAP1 column was washed with five gel-bed volumes of Gentle Ag/Ab Binding Buffer. Immunized PRAP1 rabbit serum was diluted with an equal volume of Gentle Ag/Ab Binding Buffer and added to the column and allowed it to flow through by gravity. The unbound antibodies were washed off with 5-10 gel-bed volumes of Gentle Ag/Ab Binding Buffer. The bounded antibodies were eluted by adding 510 gel-bed volumes of Gentle Ag/Ab Elution Buffer into several small fractions. Fractions were subsequently analyzed and dialyzed using a desalting column 64 (Zeba Desalt Spin Columns, 0.5mL; Pierce). Sodium azide and BSA were added to the purified antibodies and stored at 4oC. 2.8 Protein-protein interaction assay 2.8.1 Pulldown Assay Cells treated with either UV or 5-FU were lysed in lysis buffer [50mM HEPES, pH 7.4, 150mM sodium chloride, 1.5mM magnesium chlorides, 5mM EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100,1x protease inhibitor cocktail (Roche Diagnostics), 5mM sodium orthovanadate, and 25mM glycerol phosphate (Sigma)]. The glutathione S-transferase (GST)-PRAP1 proteins coupled to glutathione beads, were incubated with precleared cell lysates. The bound proteins were resolved by SDS-PAGE and were visualized by silver staining (Bio-Rad). The unique bands were excised and digested by trypsin. Mass spectra were acquired with a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer operating in the delayed-extraction reflection mode. Peptide mass fingerprints of the tryptic peptides from MALDI-TOF mass spectrometric data were used to search the National Center for Biotechnology Information protein database with the programs MS-Fit and Mascotsearch engine. This service is provided by Proteins and Proteomic Center at Department of Biological Science at NUS. 2.8.2 Immunoprecipitation Cells were plated 24 hours before transfection. One microgram of pcDNAPRAP1 plasmid was transfected per well of 6-well plate. Cells were then exposed to 5-FU. After 24 hours, cells were washed twice with PBS, lysed in IP lysis buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP40, 0.25% Na- 65 deoxycholate, and x Roche complete mini protease inhibitor cocktail) for 30 minutes on ice and collected by scraping. Lysates were centrifuged at 14,000xg for 15 minutes at 4°C and supernatants were collected. Protein concentration in the supernatant was determined using Bio-Rad protein assay kit and adjusted to 3mg/mL with lysis buffer. Samples containing 1500µg of protein were incubated with 2µg of anti-Hsp 70 or normal rabbit IgG for hours with shaking at 4°C. Protein G beads were prepared by washing with ice-cold PBS for three times and pre-cleared with lysate for 15 minutes. Immunoprecipitates were collected by the addition of pre-cleared Protein G beads and incubated for hour with shaking at 4°C. Complexes were collected by centrifugation and washed five times with lysis buffer. The pelleted beads were then resuspended in 1x SDS sample loading buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2mercaptoethanol) and heat at 95°C for minutes before analysis by SDS-PAGE and Western blotting. 2.9 Immunodetection assay 2.9.1 Immunohistochemistry Immunohistochemistry was performed using a standard indirect immunoperoxidase method. Anonymized, archived samples of human intestinal tissues were obtained from patients with surgical resection. Sections from formalin-fixed, paraffin-embedded tissues were deparaffinized by passing through three changes of xylene at minutes interval. Rehydration was performed by passing through the sections from 100% ethanol to 95% ethanol to 75% ethanol and finally to water. Antigen retrieval using the microwave heating method was 66 used. Sections were immersed in preheated antigen unmasking solution (Vectors Lab) for 10 minutes at 96oC. The sections were then treated with 3% hydrogen peroxide to remove the endogenous peroxidase. Slides were then blocked with protein block (DAKO) for 15-30 minutes. The slides were incubated with primary antibody diluted with antibody diluent (DAKO). After washing with TBST (50ml 1M Tris pH 7.4, 60.25ml of 5M NaCl, 1ml Tween 20/ 1L) for three times, slides were incubated with secondary antibody, goat anti-rabbit IgG avidine-biotinylated horseradish peroxidase conjugates (DAKO) for 15-30 minutes. The slides were then washed three times with TBST. DAB substrate was added and rinsed off after appropriate color changes were observed. The slides were then counter stained with hemotoxylin which stains the nucleus. The slides were mounted with coverslips and photographed. 2.9.2 Immunofluorescence microscopy Cells were cultured on glass coverslips in 24-well plate until the harvest time. Medium was aspirated and 300µL of 3% paraformaldehyde was added to fix the cells for 10 minutes. Fixed cells were washed twice with PBS for 10 minutes each. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. Next, the cells were blocked with blocking solution (10% FBS in PBS) for 10 minutes. Cells were washed twice with wash buffer (0.1% Triton X-100 in PBS) at minute per wash. Primary antibodies for F-actin (Phalloidin-alexa fluor 488, Molecular Probes at 1:200), tubulin (anti-tubulin monoclonal antibody, Sigma at 1:2000), PRAP1 (anti-PRAP1 polyclonal antibody at 1:1000) was added and incubated for hours at 37oC. Cells were washed two times with wash buffer. Secondary antibody (goat anti-mouse/rabbit alexa fluor 488/568) at 1:200 was 67 added for hour. Cells were washed twice with wash buffer and counter stained with Hoechst (Molecular Probes) for minutes to stain the nucleus. Cells were washed once in PBS and mounted unto slide. Images were taken using Olympus FluoView™ FV1000 at Confocal Microscopy Unit at NUS. 2.9.3 Scanning Electron microscopy Binding of PRAP1 to surface of apoptotic cells was examined by transmission electron microscopy using immunogold labeling as described in (Zou, Foong et al. 2004). Cells were exposed to sodium butyrate to induce apoptosis, fixed in 4% paraformaldehyde and incubated with anti-PRAP1 antibody. Bound antibdoy was detected with 5-nm gold particles conjugated to secondary anti-rabbit antibody. Cells were fixed in 3% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/L cacodylate for 30 minutes, followed by 2% osmium tetroxide. The cells were then dyhydrated in a graded series of methanol, transferred to acetone and dried in a in a Balzers critical-point dryer with liquefied carbon dioxide as the transition fluid. Cells were coated with 20 nm gold by means of a Balzers sputter-coater and examined with a Philips XL-30 field-emission-gun scanning electron microscope. Micrographs were taken. 2.9.4 Western blotting 2.9.4.1 Protein extraction 2.9.4.1.1 Total cell extraction At the appropriate time points, cells were harvested by trypsinization and pellet was washed once with PBS. Total protein extract was carried out using Urea lysis buffer (6M urea, 1% 2-Mercaptoethanol, 50mM Tris-HCl pH 7.4, 1% SDS 68 in PBS pH 7.4) and sonication. Lysate was centrifuged at 10,000 xg for 10 minutes at 4oC. Supernatant containing total cell lysate was transferred into a new tube. 2.9.4.1.2 Cell Fractionation For cell fractionation procedures, protease inhibitor cocktail (Roche Diagnostics) were added to the buffers. Cells were harvested and rinsed twice with cold PBS prior to lysis with detergent. Cells were resuspended in L-buffer (0.1% Triton X-100, 0.1% Nonidet P-40 in PBS, pH 7.4) and incubated on ice for 10 minutes, or until they were determined to be at least 99% lysed using trypan blue exclusion. Nuclei were then pelleted by centrifugation at 1000 x g for 10 minutes at 4oC. Supernatant was collected into another tube and classified as cytoplasm. The nuclear pellet was washed twice with L-buffer and lysed with Ubuffer [6M urea, 1% 2-mercaptoethanol, 50mM Tris buffer (pH 7.4) and 1% SDS in PBS, pH 7.4]. Nuclei were further sonicated to shear the DNA and were spun at 10000 rpm for 10 minutes at 4oC. Supernatant was collected and transferred to a new tube and was classified as nuclear lysate. 2.9.4.2 Bio-Rad protein assay Protein concentration was determined using the Bio-Rad Protein Assay Kit (Bio-Rad) with bovine serum albumin (Sigma) as the standard. One part of Dye reagent was diluted with parts of water and filtered. 10µL of protein samples at various dilutions were incubated with 200µL of diluted Dye reagent in a 96-well plate at room temperature for minutes. Absorbance was measured at 595nm using an ELISA plate reader. 69 2.9.4.3 SDS PAGE A 4% stacking gel and 12% resolving gel were casted using 30% Acrylamide/Bis Solution 37.5:1 (2.6% C). Protein lysate was mixed with 2X loading buffer and heated at 95oC for minutes and subsequently cooled on ice. Samples were then loaded into the wells of the SDS-PAGE gel and electrophoresed in SDS-PAGE buffer (3.03g Tris base, 14.4g glycine, 10mL 10% SDS/1L) at 80 V for 10 minutes, followed by 150 V for 40 minutes or until the dye front reached the end of the plate. After electophoresis, the gel was removed and equilibrated in transfer buffer (14.4g glycine, 3.03g Tris base, 10mL of 10% SDS, 20% ethanol/1L) for minutes. Proteins were transferred onto nitrocellulose (0.45µm) membrane in transfer buffer for hour at 100 V using the Bio-Rad Mini PROTEAN II (Bio-Rad). 2.9.4.4 Immunodetection After transfer, the nitrocellulose membrane was blocked with 5% non-fat milk in PBST for hour. Next, it was blocked with primary antibody diluted in 3% non-fat milk in PBST for hour at room temperature. The membrane was washed three times with PBST and then incubated with secondary antibody (Goat anti-rabbit conjugated horseradish peroxidase) at 1:10,000 dilutions in 3% non-fat milk in PBST. The blot was washed times with PBST. The bound antibodies were detected using Western Lightning chemiluminescence (Perkin Elmer) for minute. The blot was exposed to Kodak Biomax MS film (Kodak, Rochester, NY). 70 2.9.5 Immunodetection of anti-PRAP1 auto-antibody in serum For the verification of anti-PRAP1 auto-antibody in serum, 100ng of HisPRAP1 protein was loaded into each well of SDS-PAGE gel. After electrophoresis, protein was transferred unto nitrocellulose membrane and blocked with 5% non-fat milk in PBST. The blot was then incubated with serum diluted at 1:1000 in 3% non-fat milk in PBST for hour at room temperature. After three washes in PBST, the blot was incubated with secondary antibody (goat antihuman IgG conjugated-HRP) at 1:10,000 for hour at room temperature. Bounded anti-PRAP1 autoantibody was detected using Western Lightning chemiluminescence and exposed unto Kodak film for visualization of the protein band. 2.10 Bacteria binding assay 2.10.1 Detection by ELISA E.coli were chemically fixed and coated in 96-well ELISA plate overnight at 4oC. BSA coated in 96-well ELISA plate was used as negative control. The unbounded E.coli and BSA were washed with twice with PBST (PBS with 0.05% Tween-20) and blocked with 100µL assay diluent (PBS with 10% FBS, pH7) for hours at room temperature. After two washes with PBST, 50µL of HisPRAP1 or BSA or assay diluent was added and incubated for hours at room temperature. The plate was then washed four times with PBST and all the wells were incubated with 50µL of anti-PRAP1 antibody (1:5000 dilutions in assay diluent) for hour at room temperature. The plate was then washed six times with PBST and incubated with 50 µL of secondary anti-rabbit antibody conjugated-HRP (1:10,000 dilutions in assay 71 diluent) for hour at room temperature. The plate was washed six times with PBST before adding 100µL of TMB substrate buffer (dissolve tablet of TMB (Sigma) in 10mL of 0.05M phosphate citrate buffer, pH 5.0 (25.7mL of 0.2M dibasic sodium phosphate, 24.3mL of 0.1M citric acid and 50mL sterile water) with 2uL of fresh 30% H2O2). This was incubated in the dark at room temperature for 30 minutes. The reaction was stopped by adding 100µL of 1M H2SO4 to each well. Read absorbance at 450nm with reference wavelength at 570nm. 2.10.2 Detection by direct binding 2.10.2.1 Labeling of protein with Alexa Fluor dye Both BSA and HisPRAP1 were labeled with Alexa Fluor 568 using the Alexa Fluor® 594 Protein Labeling Kit (Invitrogen) according to manufacturer’s instruction. Protein was diluted to 2mg/mL in PBS. Fifty microliters of 1M bicarbonate was added to 0.5 mL of the 2mg/mL protein solution and transferred to a vial of reactive dye. The mixture was mixed by inversion and followed by stirring for hour at room temperature. Purification resin was packed into a column until the resin is about cm from the top of the column. Excess buffer was drained and reaction mixture was added to the column. The remaining reaction mixture was rinsed with 100 μL of elution buffer (0.1 M potassium phosphate, 1.5 M NaCl, pH 7.2, with mM sodium azide) and added onto the column. After the solution has entered into the column, elution buffer was slowly added into the column until the labeled protein was eluted. 2.10.2.2 Bacteria binding assay using labeled protein E.coli and Klebsiella were fixed and incubated with labeled proteins, BSA or HisPRAP1 or free dye for hours in the dark with rotation. Bacteria were spun 72 down at 13,000 rpm for minutes. The unbounded proteins or free dye were subsequently washed with PBS for three times. The reaction mixtures were then resuspended in PBS and loaded unto a 96-well plate and the fluorescence were measured using fluorescence spectrometer. 2.11 DNA damage assay 2.11.1 Alkaline single-cell gel electophoresis (comet) assay At appropriate time points, cells were harvested by trypsinization and washed once in PBS. Pellet was resuspended in ice cold HBSS solution 10% DMSO with EDTA to a density of 50-200 cells per 5µL. Six microliters of cells were mixed with 46µL of low melt agarose (0.07g in 10 mL) cooled to 39oC. The mixture was spread onto comet slide (Trevigen, Gaithersburg, MD) and kept at 4oC for it to solidify. The slide was then soaked in prechilled lysis buffer (2.5M NaCl, 0.1M EDTA pH 8, 10mM Tris base, 0.1% Triton X-100) for hour at 4oC. Slides were placed in the gel tank and were denatured with electrophoresis buffer (24g NaOH pellet, 4mL of 0.5M EDTA in 2L water) for 40 minutes in the dark. The gel was run at 25 V and 300 mA in the dark for 20 minutes. The slide was neutralized with 0.5 M Tris pH 7.4 for 15 minutes. Then the slides were dehydrated in 70% ethanol for minutes and dried at 37oC. Samples were subsequently stained with syber green solution and covered with coverslips for microscopic analysis. The tail movement was captured and analyzed using the Metasystems (Altussheim, Germany) software “Comet imager version 1.2”. One hundred randomly chosen comets were analyzed per sample. The extent of DNA damage was expressed as tail movement, which corresponds to the fraction of DNA in the tail of the comet. 73 2.11.2 Micronucleus assay At appropriate time points, cells were harvested by trypsinization and subsequently fixed using Camoy’s fixative (acetic acid/methanol, 1:3) with 3.7% formaldehyde for 10 minutes at room temperature. Cells were centrifuged at 800rpm for minutes. Fixed cells were washed three times with Camoy’s fixative. Cells were dropped onto clean slides and stained with acridine orange to differentially stain the cytoplasm and nucleus. One thousand mono-nucleated cells were scored for each sample. 2.12 Phagocytosis assay 2.12.1 Preparation of fluorescent beads Fluorescent beads were prepared as described (Arlein, Shearer et al. 1998). Caboxylate-modified 1µm yellow green fluorospheres (excitation 490nm, emission 515nm; Molecular Probes, Invitrogen) were used in in vitro phagocytosis. Before use in the phagocytosis assays, 1µL of beads were coated by incubation with 0.2 µg of proteins in PBS for hour at 4oC. 2.12.2 In vitro phagocytosis U937 cells were harvested and counted. 0.5 million cells were incubated with 0.5 µL of labeled beads (1 cell: 12 beads) or FITC-labeled E.coli in 350 µL of HBSS containing 5% FBS. In vitro phagocytosis was then carried out in the dark at 37oC with shaking at 200rpm. Cells were harvested at various time points and spun down at 1000xg for minutes at oC. The uningested beads were washed with ice-cold PBS for two times. Cells were subsequently fixed in 0.5% paraformaldehyde, filtered and analyzed by flow cytometry within minutes. 74 2.13 Statistical Analysis Nonpaired Student’s t-test was used to compare the means of two groups and p[...]... 4 .1. 1 .1 Expression of PRAP1 in intestinal epithelium 17 3 4 .1. 1 .2 PRAP1 expression is positively correlated with differentiation 17 5 4 .1. 1.3 Regulation of PRAP1 expression by differentiation 17 7 4 .1. 2 Effect of PRAP1 on differentiation 17 8 4 .1. 3 PRAP1 and innate immunity 17 9 4 .2 PRAP1, a p53-inducible modulator of cell fate in response to genotoxic stress 18 2. .. 18 2 4 .2 .1 PRAP1 is a genotoxic responsive gene 18 2 4 .2. 2 PRAP1 is a p53 responsive gene 18 4 4 .2. 3 PRAP1 modulates cell fate in response to genotoxic stress 18 6 4 .2. 4 Role of PRAP1 in cell cycle checkpoints 18 8 4.3 Role of PRAP1 in SLE 19 2 4.3 .1 Induction of PRAP1 in apoptotic cells 19 2 4.3 .2 Genotoxic agents failed to induce PRAP1 in PBMCs from SLE patients... 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... cells failed to induce prap1 gene expression 11 0 Figure 3. 42 Schematic diagram of PRAP1 gene 11 1 Figure 3.43 Schematic diagram of p53 binding site of PRAP1 gene construct 11 2 Figure 3.44 Verification of the p53 binding sites constructed plasmid 11 2 Figure 3.45 Predicted p53 binding elements of PRAP1 response to wild-type p53 11 4 Figure 3.46 Suppression of PRAP1 induction by 5-FU... apoptosis 13 7 xviii Figure 3.67 Effects of PRAP1 on actin network 14 0 Figure 3.68 Effects of PRAP1 on microtubulin network 14 2 Figure 3.69 GST-PRAP1 pull-down 14 3 Figure 3.70 Identification of PRAP1 binding protein by MS-MS 14 4 Figure 3. 71 PRAP1 physically interacts with Hsp 70 14 4 Figure 3. 72 Dying cells in floating population 14 6 Figure 3.73 Apoptotic cells in floating... 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... cell fate determination of intestinal epithelial cells and their directional migration and specific positioning (van de Wetering, Sancho et al 20 02) WNT signaling pathways play a central role in controlling the switch between proliferation and differentiation (Pinto and Clevers 20 05) in the intestinal epithelium A large number of proteins are involved in the regulation of the WNT signaling and consequent... 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 19 96), lysozyme (Mason and Taylor 19 75) and secretory phospholipase A2 (Harwig, Tan et al 19 95) 10 1. 2. 2 Structure... 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... 10 0 Figure 3. 32 Luciferase assay to identify the regions required for the prap1 gene promoter activity in HCT 11 6 cells 10 1 Figure 3.33 Identification of core promoter of prap1 gene 10 1 Figure 3.34 Induction of prap1 promoter activity by 5-FU and CPT 10 3 Figure 3.35 Induction of PRAP1 by 5-FU and CPT at mRNA level 10 4 Figure 3.36 PRAP1 was induced at protein level by 5-FU and . 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. 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. 3 .11 .1 Effect of PRAP1 on cytoskeleton 13 8 3 .11 .1. 1 Effects of PRAP1 on cellular morphology 13 8 3 .11 .1. 2 Effects PRAP1 on actin filament 13 9 3 .11 .1. 3 Effects of PRAP1 on microtubules 14 1 3 .11 .2