REGULATION AND FUNCTIONAL ANALYSIS OF THE TUMOR SUPPRESSOR GENE PRODUCT, P53. MD MONOWARUL MOBIN SIDDIQUE (M.Sc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements I am very much grateful to my supervisor A/Prof Kanaga Sabapathy, for his constant guidance, active supervision, patience and support during the course of my study and research. I thank Dr Vikhanskaya and Dr Lee Ming Kei for providing information in chapter 3.4. My sincere thanks goes to Mr Toh Wen Hong, Mr Muhammad Iqbal Dulloo, Ms Nam Shin Yuen, Ms Stephanie Soh, Ms Kelly Lum and Ms Phang Beng Hooi for their cooperation during my research. I would like to extend my gratitude to Prof Soo Khee Chee and A/Prof Hui Kam Man for providing me the opportunity to work in National Cancer Centre of Singapore. I am really grateful and indebted to my parents for all of their support that helped me to pursue my postgraduate study overseas. Md Monowarul Mobin Siddique January 2006 II TABLE OF CONTENTS TITLE PAGE I ACKNOWLEDGEMENT II TABLE OF CONTENTS III SUMMARY X LIST OF FIGURES XIV LIST OF PUBLICATIONS XVI LIST OF SYMBOLS XVII CAHPTER 1: INTRODUCTION 1.1 p53, the tumour suppressor 1.1.1 p53, the “Guardian of the Genome” 1.1.2 p53 and DNA damage response pathway 1.1.3 DNA repair and p53 1.1.4 Cell cycle arrest by p53 1.1.5 Role of p53 in DNA repair 1.1.5.1 Gadd45 activation by p53 1.1.5.2 p53R2 activation by p53 1.2 Functions of different domains of p53 1.3 Drug and chemotherapy resistance by mutant p53 11 1.4 p53 polymorphism 12 1.4.1 Distribution of p53 codon 72 polymorphism 13 1.4.2 p53 polymorphism in human malignant diseases 13 III 1.4.2.1 Breast cancer 13 1.4.2.2 Skin cancer 14 1.4.2.3 Cervical cancer 14 1.4.2.4 Gastric cancer 14 1.4.2.5 Other cancers 15 1.5 Functional difference of p53 polymorphs 16 1.5.1 Implication of p53 polymorphism in drug sensitivity along with 16 mutation 1.5.2 p53 polymorphism and breast tumour 18 1.5.3 p53 and melanin synthesis 19 1.5.4 Nucleotide excision repair by p53 22 1.5.5 p53’s role in ecological adaptation 23 CHAPTER 2: MATERIAL AND METHODS 2.1 Tissue culture 24 2.2 Transfection 24 2.3 Extraction of DNA and RNA 25 2.3.1 Extraction of genomic DNA from blood 25 2.3.2 Extraction of genomic DNA from tissue 25 2.3.3 Extraction of genomic DNA from cultured cell 25 2.3.4 Extraction of RNA from cultured cell 26 2.3.5 Extraction of RNA from blood 26 2.3.6 Extraction of RNA from tissue 26 IV 2.4 Detection of p53 codon 72 polymorphism 27 2.4.1 Purification of PCR product 28 2.4.2 Restriction digestion of PCR product (exon 2-4) to detect genotype 28 2.4.3 Sequencing of PCR product 29 2.5 One-step RT-PCR to amplify p53 gene 29 2.5.1 Sequencing of one-step RT-PCR product 30 2.6 Semi-quantitative one-step RT-PCR 31 2.6.1 Semi-quantitative PCR 31 2.6.2 Reaction condition for semi-quantitative PCR 33 2.7 Analysis of cell death 33 2.7.1 Analysis of cell cycle 34 2.8 Luciferase reporter assay 35 2.8.1 Luciferase assay 35 2.8.2 β-gal assay 36 2.8.3 Host-cell reactivation assay 36 2.9 Western blotting 37 2.10 Measuring melanin content 38 2.11 Generating p53 mutants using SDM 38 2.11.1 Primers for SDM 39 2.11.2 Generating temperature sensitive p53 40 2.12 40 Preparation of bacterial competent cell 2.12.1 Bacterial transformation 41 2.12.2 Large scale preparation of plasmid DNA 41 V 2.13 Unscheduled DNA synthesis 42 2.14 South-Western analysis 42 2.15 Micronucleus analysis 42 2.16 Immunoprecipitation 43 2.17 DNA and tissue samples 44 CHAPTER 3: RESULTS 3.1 Distribution and expression of p53 codon 72 polymorphic variants 45 3.1.1 Distribution of p53 polymorphism in Singapore 45 3.1.2 Expression of the p53 Codon 72 allele in Healthy Heterozygotes 47 3.1.2.1 Expression of p53 allele in Chinese Healthy Heterozygotes 47 3.1.2.2 Expression of p53 allele in European Healthy Heterozygotes 51 3.1.2.3 Comparison of Expression of p53 allele in Chinese and 51 European Healthy Heterozygotes 3.1.3 Expression of the p53 allele in breast cancer tissues 52 3.1.3.1 p53Arg was higher in breast cancer tissues than healthy subjects 52 3.1.3.2 Histologically normal tissues from Chinese breast cancer 53 patients preferentially express the p53Arg allele 3.1.4 Mutational status of p53 in breast cancer 53 3.2 Significance of p53 polymorphism, properties and function 59 3.2.1 Role of p53 polymorphism in DNA repair 59 3.2.2 Regulation of p53 dependent DNA-repair target gene promoters 60 by p53Pro and p53Arg variants VI 3.2.2.1 Expression of Gadd45 promoter in presence of either p53Pro 60 or p53Arg 3.2.2.2 Expression of p53R2 promoter 61 3.2.2.3 Expression of p53 target gene promoters as a control 62 3.2.3 67 p53 inducible gene expression in isogenic cell lines expressing the p53Arg or p53Pro 3.2.4 DNA repair assay using temperature sensitive-isogenic stable 67 cell lines 3.2.4.1 Apoptosis induce by temperature sensitive p53- isogenic cell lines 68 3.2.4.2 p53 dependent DNA-repair genes are differentially regulated 68 by p53Pro- and p53Argexpressing isogenic cell lines 3.2.4.3 Gadd45 expression 71 3.2.4.4 Induction of p48 by two p53 polymorphic variants 71 3.2.4.5 Ribonucleotide reductase gene, p53R2 73 3.2.5 Repair of exogenously damaged plasmids by p53 polymorphic forms 76 3.2.5.1 Host-cell reactivation assay 76 3.2.5.2 In vivo end-joining assay 81 3.2.6 Unscheduled DNA-synthesis in p53Pro and p53Arg expressing cells 81 3.2.7 Removal of cyclobutane pyrimidine dimers by p53 polymorphic 86 forms 3.2.8 Formation of micronucleic in p53Pro or p53Arg expressing cells 92 3.3 p53 polymorphism and melanin synthesis 96 VII 3.3.1 Expression of TRP-1 and Tyrosinase promoters in two p53 97 polymorphic forms 3.3.2 Synthesis of melanin in presence of two p53 polymorphic forms 99 3.3.3 Synthesis of melanin in presence of endogenous p53 103 polymorphic variants 3.4 p53 mutations and response to chemotherapeutic drugs 108 3.4.1 Generation of mutant p53 expressing isogenic cell line 108 3.4.2 Effect of p53 mutation and polymorphic status in H1299 cells to 108 anticancer drugs 3.4.3 p53 polymorphism affects the response in H1299 cells expressing 109 249-p53 to doxorubicin CHAPTER 4: DISCUSSION 4.1 Distribution and expression of p53 codon 72 polymorphism 112 4.1.1 Expression of p53 allele in healthy heterozygotes 113 4.1.2 Expression of p53 allele in breast cancer patients 114 4.1.3 Association of p53Arg allele in breast cancer susceptibility 115 4.1.4 Mutation of p53 in breast cancer 116 4.1.5 Wild-type p53Arg form may predispose to breast cancer 117 4.1.6 Expression of p53 codon 72 allele can be used as a predictive 118 factor in cancer development 4.2 Functional significance of p53 polymorphism 119 VIII 4.2.1 p53 polymorphism and DNA repair 119 4.2.2 p53Arg allele is less efficient to repair damaged DNA 121 4.2.3 Weaker property of p53Arg in DNA repair may cause cellular 121 transformation 4.2.4 Subtle difference between two polymorphs may affect p53’s 122 downstream function significantly 4.2.5 Role of p53 codon 72 polymorphism in preventing skin cancer 124 4.3 Effect of p53 polymorphism in melanin synthesis 124 4.3.1 p53Pro may protect individual from UV-induce stress 126 4.3.2 p53Arg allele predispose to skin cancer 127 4.3.3 p53 hotspot mutants along with two polymorphic forms 128 CHAPTER 5: CONCLUSION 130 REFERENCES 134 APPENDICES 164 IX SUMMARY Polymorphism at codon 72 residue of p53 results in either the p53Arg or p53Pro form of p53, whose functional significance in carcinogenesis is controversial. 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FACS analysis of cell death after treating 249-p53Pro-, 249-p53Argand vector- expressing cell lines with doxorubicin. Percentages of apoptotic cells were determined by Annexin V and propidium iodide staining. 249-p53Arg shows resistant to both doses of doxorubicin (0.25μM and 0.50μM). 165 [...]... certain types of cancer The present study was aimed to understand the association of p53 polymorphism in breast cancer in relation with the differences of mutational trend which can explain some of the differential properties of the p53 polymorphism in carcinogenesis 1.5.1 Implication of p53 polymorphism to drug sensitivity along with mutation In some cancers, the presence of specific type of p53 mutation... compared with their p53Pro-expressing counterparts (Sullivan et al., 2004), indicating that the status of the p53 codon 72 polymorphism could influence the outcome of cancer therapy Biochemical analysis has shown that 72R mutants were capable of binding to p73, the structural and functional homologue of p53, and inactivating it (Marin et al., 2000) This property of mutant p53 to bind and neutralize... whereas most of the p53Pro forms carry mutation in the DNA binding domain of p53 So, the data suggest that the expression of p53Arg allele may lead to cancer development in Asians Thus, the expression status of the p53 polymorphs, rather than the genotypic status, might be a useful indicator for cancer susceptibility Therefore, it can be predicted that p53 codon 72 polymorphism may affect p53 s function... cell cycle arrest p53 performs this function by the regulation 3 of several cellular processes and it serves as a sequence-specific transcriptional activator of genes that contain p53 responsive elements Since p53 is involved in the regulation of these distinct processes, the protein should be able to respond quickly to changes of the cell surroundings Abrogation of its function therefore leads to... activator and several functional domains have been identified within the protein The main phosphorylation domains are the N and the C terminus The N-terminal part (aa1 to aa91) contains the transcription-regulatory domain of p53, while the C-terminal domain (aa300 to aa393) controls the specific DNA binding by p53 (Steegenga et al., 1996) The C-terminal domain of p53 may 9 compete with the p53 transactivation... Mutations in p53 or in the pathway that directly it regulates have been found in over 80% of human tumors One of p53 s functions in the damage response is the activation of genes that initiates apoptosis p53 has also been shown to arrest the cell cycle in response to DNA damage, thus preventing the replication of damaged DNA These functions are mainly governed by p53 s down stream-effector genes, such... it was observed that in most of the tumors, p53 is mutated (Baker et al., 1989) and it was subsequently demonstrated that wild type p53 acts as a potent suppressor of cellular transformation It is now clear that wild type p53 is a tumor suppressor gene which prevents oncogenesis by inducing apoptosis of defected cells (Takahashi, 1989; Lane and Benchimol, 1990) The p53 tumor antigen is found in 1 increased... genotoxic stress These stresses may cause physical injury to the cell and the cells die because of the injury However, if the damage is confined to the DNA level, it is necessary for the cell to repair or fix the damage or if cannot be repaired, the defective cell should be eliminated from the body To execute these processes, cells may need to arrest cell cycle, to repair of the damaged DNA, or they may commit... etc The G1/S checkpoint prevents cells from entering the S phase in the presence of DNA damage by inhibiting the initiation of replication Under suitable conditions, cells in the G1 phase of the cell cycle become committed to enter the S phase at a stage called the restriction point in mammalian cells and start in budding yeast (Pardee, 2002) The restriction point precedes the actual start of DNA synthesis... 1.1.5.2 p53R2 activation Ribonucleotide reductase plays a key role in the synthesis of DNA and is responsible for the reduction of ribonucleotides to their corresponding deoxiribonucleotides, providing a balanced supply of precursors for DNA synthesis and repair (Wright et al., 1990; Hurta and Wright, 1992) Tanaka et al (2000) identified a novel ribonucleotide reductase gene, p53R2, a putative tumor suppressor, . REGULATION AND FUNCTIONAL ANALYSIS OF THE TUMOR SUPPRESSOR GENE PRODUCT, P53. MD MONOWARUL MOBIN SIDDIQUE (M.Sc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF. tumour suppressor 1 1.1.1 p53, the “Guardian of the Genome” 2 1.1.2 p53 and DNA damage response pathway 3 1.1.3 DNA repair and p53 3 1.1.4 Cell cycle arrest by p53 5 1.1.5 Role of p53 in. by p53 7 1.1.5.2 p53R2 activation by p53 8 1.2 Functions of different domains of p53 9 1.3 Drug and chemotherapy resistance by mutant p53 11 1.4 p53 polymorphism 12 1.4.1 Distribution of p53