C H A R A C T E R I Z A T I O N O F T H E N O V E L D O M A I N W I T H N O N A M E G E N E I N C O L O N C A N C E R Charleen Rupnarain A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science Johannesburg, 2005 i
DECLARATION I (Charleen Rupnarain) declare that this thesis is my own, unaided work. It is being submitted for the Degree of Master of Science in the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination in any other University. (Signature of candidate) day of 200 ii
PUBLICATIONS 1. Rupnarain, C., Dlamini, Z., Naicker, S. and Bhoola, K. (2004) Colon cancer: genomics and apoptotic events, Biological Chemistry, vol. 385, pp. 449-464. 2. Rupnarain, C., Dlamini, Z. and Naicker, S. RbBP6 and its gene products in colon cancer, International Immunopharmacology (submitted). 3. Rupnarain, C. and Dlamini, Z. (2004) The role of the Domain With No Name gene in colon cancer. Microscopy Society of South Africa Proceedings, Pretoria, South Africa. 4. Rupnarain, C. and Dlamini, Z. (2005) Characterization of the RbBP6 gene in colon cancer. 96 th American Association for Cancer Research Proceedings (abstract submitted). 5. Rupnarain, C. and Dlamini, Z. (2005) Characterization of the Domain With No Name Gene in colon cancer. South Africa Society of Biochemistry and Molecular Biology Proceedings, Stellenbosch, South Africa (abstract accepted). iii
DEDICATION In dedication to my family, especially my parents, and to everyone who supported me during my studies. iv
ACKNOWLEDGEMENTS I would like to extend my thanks to my supervisor, Dr Zodwa Dlamini, and my cosupervisor, Prof Sarala Naicker, for their support and guidance during my studies. My sincere thanks to Zukile Mbita for his constant help with my work. I would like to acknowledge my labmates at the Molecular and Cellular Pathology Research laboratory for their assistance with my lab work. Thank you to Prof Kanti Bhoola for his much appreciated help with my paper and my research. I would also like to thank Prof Stewart Goetsch for the tissue sections as well as for assisting me with histopathology. Thanks to Celia Snyman for the image analysis. Thanks to Rodney Hull from the Flylab for assisting me with microscope work. I would like to thank the NRF, MRC and URC for funding, as well as the University of the Witwatersrand for the Postgraduate Merit Award. v
TABLE OF CONTENTS Declaration Publications Abstract Dedication Acknowledgements List of figures List of tables Abbreviations Page ii iii iv v vi xiii xviii xix Chapter one - Introduction 1.1 The DWNN gene 1 1.1.1 The 1.1kb transcript 4 1.1.2 The 6.1kb transcript 5 1.1.3 The ubiquitin-proteasome system 6 1.1.4 DWNN homologues 11 1.1.4.1 PACT 12 1.1.4.2 RBQ-1 (RBBP6) 12 1.1.4.3 P2P-R 13 1.1.4.4 Mpe-1 protein 13 1.2 Cancer 14 1.2.1 Effects of cancer cells 14 1.2.2 Carcinogenesis 15 1.2.3 Colon cancer 16 1.2.3.1 Prevalence 16 vi
1.2.3.2 Anatomy of the colon 17 1.2.3.3 Pathology of colon cancer 18 1.2.3.4 Bacterial carcinogenesis 19 1.2.3.4.1. Escherichia coli infection 19 1.2.3.4.2 Helicobacter pylori infection 20 1.2.3.5 Aetiology 21 1.2.3.5.1 Age 21 1.2.3.5.2 Body weight 22 1.2.3.5.3 Diet 22 1.2.3.6 Induction of colon cancer 23 1.2.3.7 Genetic basis of colon cancer 25 1.2.3.8. Tumour progression 27 1.2.3.9 Key genes 31 1.2.3.9.1 APC gene 31 1.2.3.9.2 p53 tumour suppressor gene 32 1.2.3.9.3 Mdm2 33 1.2.3.9.4 Retinoblastoma gene 34 1.2.3.9.5 The deleted-in-colon-cancer gene 35 1.3 The cell cycle and tumorigenesis 35 1.3.1 p53 and Rb genes in the cell cycle 37 1.3.2 Anticancer therapies applied to the cell cycle 38 1.4 Apoptosis 39 1.4.1 Apoptosis and cancer 40 1.4.2 Pathways of apoptosis 41 1.4.2.1 Caspases 41 vii
1.4.2.2 The intrinsic/mitochondrial pathway 43 1.4.2.3 The extrinsic/death-receptor pathway 45 1.4.3 Bcl-2 family 47 1.4.3.1 PUMA 50 1.4.4 Tumour necrosis factor family 51 1.4.4.1 TRAIL 51 1.4.4.2 Fas ligand and Fas receptor 51 Chapter two Methods and Materials 2.1 Materials 53 2.2 Methods: RNA extraction 53 2.3 Quantitative PCR 54 2.3.1 Control 57 2.4 In situ hybridisation 57 2.4.1 Probe preparation 57 2.4.1.1 Ligation and transformation 57 2.4.1.2 Miniprep 59 2.4.1.3 Restriction digestion 61 2.4.1.4 Isolating band from gel 61 2.4.1.5 Dissolving the gel slice 62 2.4.1.6 DNA purification 62 2.4.1.7 Spectrophotometry 63 2.4.1.8 Digoxigenin labelling 64 2.4.1.9 Estimation of minimal probe concentration 66 2.4.1.10 Concentration estimation for the probe 67 viii
2.4.2 In situ hybridisation 68 2.4.2.1 Pre-hybridisation 68 2.4.2.2 Hybridisation 69 2.4.2.3 Post-hybridisation and detection 69 2.4.2.4 Colorimetric ISH 70 2.4.2.5 Fluorimetric ISH 71 2.4.2.6 Controls 71 2.5 Immunocytochemistry (DWNN) 72 2.5.1 Controls 73 2.5.2 Statistical analysis 73 2.6 Immunocytochemistry (Helicobacter pylori) 74 2.6.1 Controls 74 2.7 TUNEL 75 2.7.1 Controls 76 2.8 Proliferation assay 77 2.8.1 Controls 78 2.9 Bcl-2 assay 79 2.9.1 Controls 79 Appendix 1 Reagents and Solutions 80 Chapter three - Results 3.1 Histopathology 88 3.1.1 Introduction 88 3.1.2 Normal colon tissue 88 3.1.3 Tumour classification 89 ix
3.1.4 Summary 94 3.2 Quantitative PCR 95 3.2.1 Summary 96 3.3 In situ hybridisation 97 3.3.1 Probe synthesis of the 1.1kb transcript 97 3.3.1.1 Ligation and transformation 97 3.3.1.2 DNA isolation and linearisation 98 3.3.1.3 Digoxigenin labelling of probe 100 3.3.1.4 Colorimetric ISH images 101 3.3.1.5 FISH images 105 3.3.1.6 Summary 108 3.3.2 In situ hybridisation of the 6.1kb + E16 mrna 109 3.3.2.1 Probe synthesis 109 3.3.2.2 Colorimetric ISH images 109 3.3.2.3 FISH images 112 3.3.2.4 Summary 114 3.3.3 In situ hybridisation of the exon 16 mrna 115 3.3.3.1 Colorimetric ISH images 115 3.3.3.2 Summary 117 3.4 Immunocytochemistry (DWNN) 118 3.4.1 Localisation of the DWNN protein 119 3.4.2 Localisation of the RBBP6 protein 124 3.4.3 Image analysis of the DWNN protein 127 3.4.4 Summary 129 3.5 TUNEL 130 x
3.5.1 TUNEL images 131 3.5.2 Summary 134 3.6 Ki-67 proliferation assay 135 3.6.1 Ki-67 images 135 3.6.2 Summary 137 3.7 Bcl-2 assay 138 3.7.1 Bcl-2 images 138 3.7.2 Summary 140 3.8 Helicobacter pylori localisation 141 3.8.1 H. pylori ICC images 141 3.8.2 Summary 144 Chapter four - Discussion 4.1 Colon cancer 145 4.2 DWNN 147 4.3 Quantitative PCR 149 4.4 In situ hybridisation 150 4.5 Immunocytochemistry 152 4.6 TUNEL 154 4.7 Ki-67 proliferation assay 155 4.8 Bcl-2 assay 157 4.9 Helicobacter pylori localisation 158 xi
Chapter five Conclusion 160 References 162 xii
LIST OF FIGURES Page Figure 1.1: Conserved amino acids within the DWNN domain in multiple species 2 Figure 1.2: Domain structure of the DWNN protein in different species 3 Figure 1.3: Structure of the DWNN domain making up DWNN-13 4 Figure 1.4: Protein translation of the DWNN-13 gene 4 Figure 1.5: DWNN-200 domain structure showing the protein without and with alternative splicing 6 Figure 1.6: The DWNN proteins and the DWNN partial cdnas 11 Figure 1.7: The initiation of cancer 24 Figure 1.8: The genetic changes involved in colon cancer development 30 Figure 1.9: The intrinsic apoptotic pathway 44 Figure 1.10: The extrinsic apoptotic pathway 46 Figure 3.1: Normal colonic tissue 89 Figure 3.2: Poorly differentiated adenocarcinoma 90 Figure 3.3: Moderately differentiated adenomatous glands 91 Figure 3.4: Well differentiated adenocarcinoma 91 Figure 3.5: Well differentiated adenocarcinoma in smooth muscle 92 Figure 3.6: Well differentiated adenocarcinoma in muscularis propria 93 Figure 3.7: Moderately differentiated adenomatous glands in the submucosa 93 Figure 3.8: Well differentiated adenomatous glands 94 Figure 3.9: Amplification of the DWNN transcripts via real-time PCR in a normal kidney cell-line and a colon cancer cell-line 96 Figure 3.10: Agarose gel showing restriction digestion 99 Figure 3.11: Diagrammatic representation of in situ hybridisation 100 Figure 3.12: Negative control 101 xiii
Figure 3.13: Normal colon tissue 101 Figure 3.14: Localisation of the 1.1kb mrna in the subserosa 102 Figure 3.15: Localisation of the 1.1kb mrna in lymphocytes 102 Figure 3.16: Localisation in moderately differentiated adenocarcinoma 103 Figure 3.17: Localisation in muscularis mucosae 103 Figure 3.18: Nuclear localisation of the 1.1kb mrna 104 Figure 3.19: Localisation in dysplastic lamina propria 104 Figure 3.20: Negative control 105 Figure 3.21: Localisation in lymphocytes 105 Figure 3.22: Cytoplasmic localisation of the 1.1kb mrna 106 Figure 3.23: Localisation in moderately differentiated adenocarcinoma 106 Figure 3.24: Localisation in muscularis propria 107 Figure 3.25: Localisation in the submucosa 107 Figure 3.26: Normal colon tissue 109 Figure 3.27: Nuclear localisation of 6.1kb + E16 mrna 110 Figure 3.28: Localisation in moderately differentiated adenocarcinoma 110 Figure 3.29: Cytoplasmic and nuclear localisation 111 Figure 3.30: Localisation of 6.1kb + E16 mrna in lymphocytes 111 Figure 3.31: Negative control 112 Figure 3.32: Cytoplasmic localisation of 6.1kb + E16 mrna 112 Figure 3.33: Nuclear and cytoplasmic localisation of 6.1kb + E16 mrna 113 Figure 3.34: Cytoplasmic localisation 113 Figure 3.35: Minimal localisation of 6.1kb + E16 mrna 114 Figure 3.36: Normal colon tissue 115 Figure 3.37: Nuclear localisation of the exon 16 mrna 116 xiv
Figure 3.38: Cytoplasmic and nuclear localisation of exon 16 mrna 116 Figure 3.39: Localisation in lymphocytes 117 Figure 3.40: Diagrammatic representation of the reactions involved in immunocytochemistry 119 Figure 3.41: Negative control 119 Figure 3.42: Positive control (testes) 120 Figure 3.43: Normal undiseased colon 120 Figure 3.44: Cytoplasmic localisation of the DWNN protein 121 Figure 3.45: Cytoplasmic localisation of the DWNN protein in lymphocytes 121 Figure 3.46: Nuclear and cytoplasmic localisation 122 Figure 3.47: Crypts of Lieberkühn 122 Figure 3.48: Blood vessel 123 Figure 3.49: Normal colon tissue 124 Figure 3.50: Cytoplasmic localisation of the RBBP6 protein 124 Figure 3.51: Localisation in well differentiated adenomatous glands 125 Figure 3.52: Localisation in moderately differentiated adenocarcinoma 125 Figure 3.53: Localisation in the muscularis propria 126 Figure 3.54: Localisation in well differentiated adenocarcinoma 126 Figure 3.55: Average immunolabelling of DAB per area 127 Figure 3.56: Diagrammatic representation of reactions occurring in TUNEL 131 Figure 3.57: Normal undiseased tissue 131 Figure 3.58: Nuclear labelling in poorly differentiated adenocarcinoma 132 Figure 3.59: Nuclear and cytoplasmic labelling 132 Figure 3.60: Nuclear labelling in moderately differentiated adenocarcinoma 133 Figure 3.61: Nuclear and cytoplasmic labelling in necrotic debris 133 xv
Figure 3.62: Cytoplasmic and nuclear proliferation 135 Figure 3.63: Nuclear staining 136 Figure 3.64: Nuclear localisation in moderately differentiated adenocarcinoma 136 Figure 3.65: Ki-67 localisation 137 Figure 3.66: Bcl-2 localisation 138 Figure 3.67: Bcl-2 localisation in moderately differentiated adenocarcinoma 139 Figure 3.68: Nuclear localisation of Bcl-2 139 Figure 3.69: Normal colon tissue 141 Figure 3.70: Localisation in lymphocytes 142 Figure 3.71: H. pylori localisation in poorly differentiated adenocarcinoma 142 Figure 3.72: Cytoplasmic localisation in lumen 143 Figure 3.73: Localisation of H. pylori in well differentiated adenocarcinoma 143 xvi
LIST OF TABLES Page Table 1.1: Lifetime risks of colorectal cancers per population group in South Africa (1993-1995) 16 Table 2.1: cdna synthesis (RT) 55 Table 2.2: Cocktail for RT-PCR 56 Table 2.3: Reactions for RT-PCR 56 Table 2.4: Cocktail for ligation 58 Table 2.5: Restriction digestion 61 Table 2.6: Labelling with Digoxigenin 64 Table 2.7: Dilutions of labelled probes 66 xvii