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

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1 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

2 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

3 PUBLICATIONS 1. Rupnarain, C., Dlamini, Z., Naicker, S. and Bhoola, K. (2004) Colon cancer: genomics and apoptotic events, Biological Chemistry, vol. 385, pp 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

4 DEDICATION In dedication to my family, especially my parents, and to everyone who supported me during my studies. iv

5 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

6 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 The 1.1kb transcript The 6.1kb transcript The ubiquitin-proteasome system DWNN homologues PACT RBQ-1 (RBBP6) P2P-R Mpe-1 protein Cancer Effects of cancer cells Carcinogenesis Colon cancer Prevalence 16 vi

7 Anatomy of the colon Pathology of colon cancer Bacterial carcinogenesis Escherichia coli infection Helicobacter pylori infection Aetiology Age Body weight Diet Induction of colon cancer Genetic basis of colon cancer Tumour progression Key genes APC gene p53 tumour suppressor gene Mdm Retinoblastoma gene The deleted-in-colon-cancer gene The cell cycle and tumorigenesis p53 and Rb genes in the cell cycle Anticancer therapies applied to the cell cycle Apoptosis Apoptosis and cancer Pathways of apoptosis Caspases 41 vii

8 The intrinsic/mitochondrial pathway The extrinsic/death-receptor pathway Bcl-2 family PUMA Tumour necrosis factor family TRAIL Fas ligand and Fas receptor 51 Chapter two Methods and Materials 2.1 Materials Methods: RNA extraction Quantitative PCR Control In situ hybridisation Probe preparation Ligation and transformation Miniprep Restriction digestion Isolating band from gel Dissolving the gel slice DNA purification Spectrophotometry Digoxigenin labelling Estimation of minimal probe concentration Concentration estimation for the probe 67 viii

9 2.4.2 In situ hybridisation Pre-hybridisation Hybridisation Post-hybridisation and detection Colorimetric ISH Fluorimetric ISH Controls Immunocytochemistry (DWNN) Controls Statistical analysis Immunocytochemistry (Helicobacter pylori) Controls TUNEL Controls Proliferation assay Controls Bcl-2 assay Controls 79 Appendix 1 Reagents and Solutions 80 Chapter three - Results 3.1 Histopathology Introduction Normal colon tissue Tumour classification 89 ix

10 3.1.4 Summary Quantitative PCR Summary In situ hybridisation Probe synthesis of the 1.1kb transcript Ligation and transformation DNA isolation and linearisation Digoxigenin labelling of probe Colorimetric ISH images FISH images Summary In situ hybridisation of the 6.1kb + E16 mrna Probe synthesis Colorimetric ISH images FISH images Summary In situ hybridisation of the exon 16 mrna Colorimetric ISH images Summary Immunocytochemistry (DWNN) Localisation of the DWNN protein Localisation of the RBBP6 protein Image analysis of the DWNN protein Summary TUNEL 130 x

11 3.5.1 TUNEL images Summary Ki-67 proliferation assay Ki-67 images Summary Bcl-2 assay Bcl-2 images Summary Helicobacter pylori localisation H. pylori ICC images Summary 144 Chapter four - Discussion 4.1 Colon cancer DWNN Quantitative PCR In situ hybridisation Immunocytochemistry TUNEL Ki-67 proliferation assay Bcl-2 assay Helicobacter pylori localisation 158 xi

12 Chapter five Conclusion 160 References 162 xii

13 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

14 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

15 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

16 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 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

17 LIST OF TABLES Page Table 1.1: Lifetime risks of colorectal cancers per population group in South Africa ( ) 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