Telomere chromosome integrity in breast cancer

ACKNOWLEDGEMENTS It is a pleasure to thank many people who made this thesis possible. First and foremost, I would like to express my most sincere thanks to my mentor and supervisor, Associate Professor M. Prakash Hande for his patience, encouragement, unfailing guidance and sound advice throughout my candidature. His ideas and constructive criticisms contributed to the overall caliber of the thesis. I am very grateful to him for giving me an opportunity to work under his supervision and introducing me to the real world of research. I thank Dr. Razq Hakem, Associate Professor, Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, Toronto, Canada for fruitful collaboration with us on Brca1 mouse studies. Dr. J. Peter McPherson, Assistant Professor, Department of Pharmacology, University of Toronto, Toronto, Canada is acknowledged for sharing the data generated from Brca1 knockout mice. Thanks to Dr. Thomas Choudary Putti and Dr. Rajiv Singh from the Department of Pathology, National University Hospital, Singapore, for providing the tissue samples, without which this study would not be completed. Special thanks to Dr. Nallasivam Palanisamy and Ms. Kalpana Ramnarayanan from Genome Institute of Singapore for guiding me in array-CGH and letting us use their facilities. I am immensely thankful to my dear friend, Ms. Lakshmidevi Balakrishnan with whom I shared many cherished moments and has given me moral support whenever I needed. I would also like to thank Dr. Birendranath Banerjee with whom I have i shared many scientific discussions during the course of work. Heartfelt thanks to Dr. Vinoth Kumar Jayaseelan, Mr. Resham Lal Gurung and Dr. Swaminathan Sethu for extending their support on many occasions. I would, in addition, like to thank my lab mates in Genome stability lab, Mr. Aik Kia Khaw, Ms. Grace Low Kah Mun, Mr. Aloysius Ting Poh Leong, Ms. Inthrani Rajainthran, Ms. Asha Rani for their help. My special thanks to Ms. Yasaswini Sampath Kumar and Mr. Dulesh Pieris for their help on many occasions. I would also like to thank all my friends at the Department of Physiology, who helped me in one or another way during my stay in the department. My special thanks to my friend, Dr. Prathiba Kurupati for guiding me in RT-PCR. This work was supported by grants from Biomedical Research Council of Singapore. Finally, I would like to specially thank my husband and daughter for their constant support and enormous encouragement which was invaluable in solving many challenges which arose. Without their steady support this work would never have been accomplished. I am also very grateful to my parents for their continuous moral support. ii TABLE OF CONTENTS ACKNOWLEDGMENTS………………………………………… i TABLE OF CONTENTS…………………………………………….iii SUMMARY………………………………………………………… viii LIST OF TABLES …………………………………………………x LIST OF FIGURES……………………………………………… xi ABBREVIATIONS………………………………………………… .xiv LIST OF PUBLICATIONS………………………………………… xv CHAPTER 1. INTRODUCTION 1.1: Telomeres 1.1.1: Telomeric structure 1.1.2: Regulation of telomere function 1.1.2.1: Telomeric end-replication problem 1.1.2.2: Regulation of telomere length by telomerase 10 1.1.2.3: Regulation of telomeres by telomere binding proteins 12 1.1.2.4: DNA repair proteins in telomere maintenance 15 1.2: Breast cancer susceptibility gene (BRCA1) 20 1.2.1: Functions of BRCA1 22 1.2.1.1: Role of BRCA1 in DNA repair 23 1.2.1.2: Role of BRCA1 in transcriptional regulation 24 iii 1.2.1.3: Role of BRCA1 in cell cycle control 26 1.2.1.4: Role of BRCA1 in ubiquitination 26 1.2.1.5: Role of BRCA1 in development 28 1.3: Telomere mediated genomic instability in cancer 29 1.4: Breast Cancer 33 1.4.1: Types of Breast Cancer 33 1.4.2: Risk Factors for breast cancer 34 1.4.3: Prognosis of breast cancers 36 1.5: Hypothesis 38 1.6: Objectives 39 1.7: Significance 39 CHAPTER 2. MATERIALS AND METHODS 41 2.1: Role of BRCA1 in telomere maintenance 41 2.1.1: Peripheral activated T-cells from tBrca1–/– mice 41 2.1.2: Quantitative fluorescence in situ hybridization (qFISH) 41 2.1.3: Spectral Karyotyping (SKY) analysis 43 2.1.4: Chromosome immuno-fluorescence 43 2.1.5: Immunofluorescence with PNA-FISH 44 2.1.6: Colocalisation studies 45 2.2: Role of telomere dysfunction in breast cancer progression 45 2.2.1: Patient Samples 45 2.2.1.1: Histopathological grading 47 iv 2.2.2: Breast cancer cell lines 48 2.2.3: Telomere length measurement by southern blotting telomere restriction fragments 49 2.2.4: Peptide Nuclei Acid Fluorescence In situ hybrididation (PNA FISH) to measure telomere length on tissue sections 50 2.2.5: Anaphase bridges and inter-nuclear bridges 51 2.2.6: Cytokinesis block micronucleus assay (CBMN assay) 51 2.2.7: Peptide Nucleic Acid – Fluorescence in situ Hybridisation (PNA FISH) on cell lines for chromosomal analysis 51 2.2.8: Multicolour fluorescence in-situ hybridization (mFISH) on cell lines for analyzing chromosomal aberrations 52 2.2.9: Array comparative genomic hybridization (Array-CGH) on selected tissue samples 53 2.3: Association of telomere dysfunction with telomere regulators 56 in breast cancer 2.3.1: Telomerase activity by telomere repeat amplification protocol assay 56 (TRAP) 2.3.2: Real time Quantitative Reverse Transcriptase-PCR (RT-PCR) for hTERT 57 expression 2.3.3: Real-Time Quantitative RT-PCR for telomere related genes 57 CHAPTER 3. RESULTS 59 3.1: Role of BRCA1 in telomere maintenance 59 3.1.1: Telomere shortening in Brca1 deficient peripheral activated T cells 59 3.1.2: Telomere shortening is associated with increased aneuploidy and chromosomal end to end fusions in tBrca1-/- peripheral T cells 62 3.1.3: PNA FISH analysis of tBrca1-/-p53-/- tumours showed 65 v chromosomal abnormalities indicative of telomere dysfunction 3.1.4: Structural and numerical chromosomal changes revealed by SKY analysis of tBrca1–/–p53–/– tumours 65 3.1.5: BRCA1 partially colocalises with the telomeres and TRF2 67 3.2: Role of telomere dysfunction in breast cancer progression 73 3.2.1: Telomere shortening in breast cancer tissues with greater shortening in the higher grades of tumours 73 3.2.2: Telomere shortening observed in breast cancer cell lines compared to normal cell lines 76 3.2.3: Telomere shortening associated with increased chromosomal instability 77 3.2.4: Increased chromosomal instability in breast cancer cell lines compared to normal cells 79 3.2.5: Increased genomic instability in the form of amplifications or deletions in higher grades of breast cancer by Array CGH 90 3.3: Association of telomere dysfunction with telomere length regulators in breast cancer 99 3.3.1: hTERT expression increases from premalignant to malignant cells 99 3.3.2: Higher Telomerase activity in breast cancer samples than the adjacent tissues 101 3.3.3: Differential expression of mRNA for TRF1, POT1 and TANKYRASE1 in association to the histological grades of the tumours 102 CHAPTER 4. DISCUSSION 108 4.1: Telomere mediated-genomic instability in tumourigenesis 108 4.2: Role of BRCA1 in telomere maintenance 108 4.3: Role of telomere dysfunction in breast cancer progression 112 vi 4.4: Association of telomere dysfunction with telomere length regulators in breast cancer 118 CHAPTER 5. CONCLUSION 124 CHAPTER 6. REFERENCES 127 CHAPTER 7. APPENDIX 152 7.1: List of selected amplified genes in different grades of breast cancer 152 7.1.1: List of the amplified genes in the grade II tumours, involved 152 in the function of signal transduction 7.1.2: List of the amplified genes in grade III tumours, involved 153 in the function of signal transduction 7.1.3: List of the amplified genes in the grade II tumours, involved 155 in the regulation of cellular processes 7.1.4: List of the amplified genes in the grade III tumours, involved 157 in the regulation of cellular processes 7.2: List of selected deleted genes in different grades of breast cancer 165 7.2.1: List of the deleted genes in the grade II tumours, involved 166 in the regulation of cellular processes 7.2.2: List of the deleted genes in the grade III tumours, involved 167 in the regulation of cellular processes vii SUMMARY Genomic instability is the hallmark of cancer and it is involved in multistep tumour progression. Telomere integrity is one of the critical factors in maintaining genomic stability. In addition to telomerase and telomere related proteins, a number of DNA repair proteins are increasingly discovered to play a role in telomere integrity. BRCA1 has been shown to interact with DNA repair proteins like ATM, Rad50, MRN complex which have an established role in telomere maintenance. Brca1-/- T-cells derived from Brca1 conditional knockout mice under either p53-/- or Bcl2 background displayed telomere dysfunction as both losses of telomere repeats as well as defective telomere capping. Karyotyping of tBrca1-/- p53-/- thymomas revealed the presence of clonal chromosomal translocations containing telomeric DNA at the fusion points suggestive of the contribution of telomeric dysfunction. BRCA1 also showed partial recruitment to the chromosomal ends and partial colocalisation with TRF2 which increased with Bleomycin treatment. The results thus provide evidence that BRCA1 has a role in maintaining telomere integrity. To further explore the association between telomere-mediated instability and tumour progression, breast cancer was studied as a model. Analysis of archived human breast tumor tissues revealed significantly greater telomere shortening and associated telomeric dysfunction in higher grade tumours (p[...]... genomic instability in breast tumour cells and in cells from mice lacking breast cancer genes 66th Annual Meeting of the Japanese Cancer Association October 3-5, 2007, Pacifico Yokohama, Yokohama, Japan 2 Poonepalli A, Banerjee B, Tat Xin Ee, Putti T, McPherson JP, Hakem R, Hande MP Telomere- mediated genomic instability in breast tumour cells and in cells from mice lacking breast cancer genes International... telangiectasia mutaed BRCA1-binding protein Breakage–fusion–bridge cycles Breast cancer susceptibility gene1 Brca1 C-Terminal 4,6-diamino-2-phenylindole Ductal carcinoma in situ DNA-dependent protein kinase catalytic subunit Oestrogen receptor Fluorescence in- situ hybridisation Fluorescein isothiocyanate Hormone receptor human repressor activator protein 1 Invasive ductal carcinoma Mouse embryonic fibroblasts... enzyme TRF-interacting protein POT1 interacting protein 1 Texas red Telomere repeat binding factor 1 Telomere repeat binding factor 2 xiv LIST OF PUBLICATIONS 1 Choong LY, Lim S, Loh MC, Man X, Chen Y, Toy W, Pan M, Chen CS, Poonepalli A, Hande MP, Tan PH, Salto-Tellez M, Wong CY, Shah N, Druker BJ, Lim YP Progressive loss of epidermal growth factor receptor in a subpopulation of breast cancers: implications... TRF2 in Hela cells treated with 5µg/ml of Bleomycin 72 Figure 19 Average percentage decrease in telomere length in different grades of tumour tissues (n=46) 74 Figure 20 Telomere length measured on paraffin tissue sections by PNAFISH 75 Figure 21 Telomere length measured in breast cancer cell lines by southern blotting analysis of telomere restriction fragments 76 Figure 22 Anaphase bridges as an indicator... 2005 Oxidative damage-induced cell death, telomere attrition and genomic instability in mouse cells lacking PARP-1.Cold Spring Harbor Laboratory Meeting on Telomeres and Telomerase, May 4 to May 8, 2005, page 116 7 P.Anuradha, Aik Kia Khaw, Adayabalam S Balajee, M Prakash Hande Oxidative stress-Induced Cell Death, Telomere Attrition and Genomic Instability in Mouse cell lines Lacking Poly (ADP-ribose)... from mice lacking breast cancer genes International Syposium on genomic instability and cancer July 22-26, 2007, Srinagar, J&K, India 3 Poonepalli A, McPherson JP, Banerjee B, Khaw A, Putti T, Hakem R, Hande MP Telomere- mediated genomic instability in breast tumour cells and in xvi cells from mice lacking breast cancer genes 11th International Congress of Human Genetics , Brisbane, August 6-10, Australia,... R, Jasin M, Hande MP, Hakem R( 2004 ) Involvement of Mammalian Mus81 in Genome Integrity and Tumor Suppression Science 304 (5678): 1822-1826 8 Poonepalli A et al., (2008) Telomere mediated genomic instability correlates with the clinico-pathological parameters of breast cancer (Manuscript submitted) CONFERENCES: 1 Hande MP, Poonepalli A, Banerjee B, Tat Xin Ee, Putti T, McPherson JP, Hakem R Telomere- mediated... Anaphase bridges as an indicator of telomere dysfunction 78 Figure 23 PNA FISH analysis using Cy3 labelled telomere specific probe and FITC labeled centromere specific probe 81 Figure 24 Micronucleus analysis of Breast cancer cell lines 82 Figure 25 mFISH analysis of breast cancer cell lines 85 Figure 26 Complete genome view by array-CGH showing greater genomic instability in the higher grade than the lower... Chromosome integrity in XP-A fibroblasts 3rd Asia Pacific Anti-ageing Conference & Exhibition 2004 10 Miranti Silasudjana, Aik Kia Khaw, Anuradha Poonepalli, Cao jie, M.Prakash Hande Effect of Histone deacetylase inhibitor on telomere integrity and Telomerase in Human tumour cells 3rd Asia Pacific Anti-ageing Conference & Exhibition 2004 11 P Anuradha, Sun XL, Venkat A, Wong YC, Gole LA.Fluorescence in- situ... November 17, 2005 (Invited Presentation).Oxidative damage-induced telomere attrition and genomic instability in DNA repair deficient mammalian cells 5 Newman JPA, Balakrishnan L, Khaw Ak, Poonepalli A, Lee H-W, Hande MP 2005 Short dysfunctional telomeres impair the repair of arsenite-induced oxidative damage in mouse cells, Keystone Symposium on Stem Cells, Senescence and Cancer, Singapore (Oct 2005) . with the telomeres and TRF2 67 3.2: Role of telomere dysfunction in breast cancer progression 73 3.2.1: Telomere shortening in breast cancer tissues with greater shortening 73 in the higher. 3.2.2: Telomere shortening observed in breast cancer cell lines compared 76 to normal cell lines 3.2.3: Telomere shortening associated with increased chromosomal instability 77 3.2.4: Increased. Genomic instability is the hallmark of cancer and it is involved in multistep tumour progression. Telomere integrity is one of the critical factors in maintaining genomic stability. In addition