Analysis of NEIL3 Expression: A Possible Resistance Factor to Cancer Chemotherapy in Paediatric Cancer Cells. Amira Duweb School of Environment & Life Sciences. University of Salford, Salford, UK A thesis submitted for the degree of MSc by Research, 2015 Table of Contents Table of Contents.........................................................................................................2 List of Abbreviations...................................................................................................6 Abstract........................................................................................................................10 Declaration...................................................................................................................11 Acknowledgments……………………………………………………………………11 Chapter 1: Introduction……………………………………………………………..12 1.1 Cancer……………………………………………………………………....12 1.1.1 Childhood cancer...................................................................................12 1.1.2 Leukaemia..............................................................................................13 1.1.2.1 Acute lymphoblastic leukaemia.................................................14 1.1.2.1 (i) The molecular genetic aspects of acute lymphoblastic leukaemia …………………………………………..15 1.1.2.1 (ii) The effect of glucocorticoids on acute lymphoblastic leukaemia…………………………………………..18 1.1.3 Central nervous system tumours............................................................19 1.1.3.1 Medulloblastoma………………………………………………20 1.1.3.1 (i) The molecular genetic aspects of medulloblastoma.20 1.1.4 Bone tumours..........................................................................................22 1.1.4.1 Osteosarcoma…………………………………………………..22 1.1.4.1 (i) The molecular genetic aspects of osteosarcoma….. 23 1.1.5 Hepatic tumours......................................................................................25 1.1.5.1 Hepatoblastoma………………………………………………..25 1.1.5.1 (i) The molecular genetic aspects of hepatoblastoma...26 1.2 DNA damage..................................................................................................27 1.2.1 Aetiology of damaged DNA…………………………………………...27 1.2.2 Reactive oxygen species ………………………………………………29 2 1.3 Types of DNA repair mechanisms………………………………………31 1.3.1 Direct reversal……………………………………………………….31 1.3.2 Nucleotide excision repair ………………………………………….32 1.3.3 Double-strand break repair …………………………………………32 1.3.4 Mismatch repair …………………………………………………….33 1.3.5 Base excision repair ………………………………………………...33 1.4 Oxidized base-specific DNA glycosylases in eukaryotic cells………....35 1.5 NEIL family………………………………………………………………36 1.5.1 NEIL1………………………………………………………………...36 1.5.2 NEIL2………………………………………………………………...37 1.5.3 NEIL3………………………………………………………………...38 1.5.3.1 Analysis of the structure of NEIL3 and its DNA glycosylase activity………………………………………………………..39 1.5.3.2 The role of NEIL3 in rapidly dividing cells………………….41 1.5.3.3 The role of NEIL3 in replication-associated repair…………42 1.5.3.4 The immunological role of NEIL3…………………………...43 1.5.3.5 NEIL3 and human immunodeficiency virus type1…………..43 1.6 Telomeres………………………………………………………………….44 1.6.1 Factors that influence telomere length……………………………….45 1.6.2 Factors regulating telomere homeostasis…………………………….47 1.6.3 Telomerase…………………………………………………………...48 1.6.4 The association between telomeric proteins and DNA repair mechanisms………………………………………………………….51 1.7 Genotoxic drugs…………………………………………………………..52 1.7.1 Cisplatin……………………………………………………………...53 1.7.1.1 Pharmacokinetic properties of cisplatin……………………..54 1.7.1.2 Pharmacodynamic properties of cisplatin…………………...54 1.7.1.3 Mechanisms of cisplatin resistance………………………….55 1.7.2 tert-butyl hydroperoxide…………………………………………….56 1.7.2.1 Pharmacokinetic properties of tert-butyl hydroperoxide…....56 3 1.7.2.2 Pharmacodynamic properties of tert-butyl hydroperoxide….57 1.7.3 Doxorubicin………………………………………………………….57 1.7.3.1 Pharmacokinetic properties of doxorubicin…………………59 1.7.3.2 Pharmacodynamic properties of doxorubicin ……………….59 1.7.3.3 Mechanisms of doxorubicin resistance……………………....61 Aims of the project………………………………………………………………….62 Chapter 2: Materials and Methods………………………………………………..63 2.1 Materials…………………………………………………………………..63 2.1.1 Chemicals…………………………………………………………….63 2.1.2 Kits and other lab consumables……………………………………...64 2.1.3 PCR Primers…………………………………………………………65 2.2 Methods …………………………………………………………………...66 2.2.1 Cell culture…………………………………………………………...66 2.2.1.1 Cell lines……………………………………………………...66 2.2.1.2 Growing and maintaining the suspension cells……………....67 2.2.1.3 Splitting the suspension cells…………………………………67 2.2.1.4 Growing and maintaining the adherent cells…………………67 2.2.1.5 Splitting the adherent cells…………………………………….68 2.2.1.6 Freezing the suspension cells and the adherent cells ………....68 2.2.1.7 Thawing the suspension cells and the adherent cells………….68 2.2.1.8 Cell counting…………………………………………………..69 2.2.1.9 Time course of cell growth…………………………………….69 2.2.1.10 Preparation cell pellets for RNA extraction ………………….69 2.2.2 RNA extraction………………………………………………………..69 2.2.3 Reverse transcription…………………………………………………..71 2.2.4 Reverse transcription PCR…………………………………………….72 2.2.5 Quantitative real time PCR……………………………………………76 2.2.6 Cytotoxicity assays…………………………………………………….78 2.2.6.1 MTS assay……………………………………………………..78 2.2.6.2 MTT assay……………………………………………………..81 2.2.7 qRT-PCR on treated HepG2 cells……………………………………..82 4 2.2.7.1 Preparation of 2.5 µM and 5 µM tert-butyl hydroperoxide solution………………………………………………………82 Chapter 3: Results………………………………………………………………….84 3.1 Identification of the exponential growth phase of each cell line………….84 3.2 RNA extraction from the cancer cell lines………………………………...86 3.3 Specificity of the PCR primers……………………………………………89 3.4 Quantification of gene expression by qRT-PCR…………………………..91 3.5 Determination the cell growth inhibitory effect of genotoxic agents using the MTS assay………………………………………………………97 3.6 Determination the cell growth inhibitory effect of genotoxic agents using the MTT assay………………………………………………………100 3.7 Estimation of the effect of tert-butyl hydroperoxide on HepG2 cells on target gene expression by qRT-PCR……………………………...101 Chapter 4: Discussion ……………………………………………………………...103 4.1 The significant role of NEIL3……………………………………………...103 4.2 The effect of oxidative stress on steroid sensitive and steroid resistant acute lymphoblastic leukaemia…………………………………………………..105 4.3 The effect of oxidative stress on hepatoblastoma cells……………………..106 4.4 The effect of cisplatin on steroid sensitive, steroid resistant acute lymphoblastic leukaemia and hepatoblastoma cells……………………….107 4.5 The effect of doxorubicin on steroid sensitive, steroid resistant acute lymphoblastic leukaemia and hepatoblastoma cells………………………107 Conclusion…………………………………………………………………………..108 Future perspectives………………………………………………………………....108 References…………………………………………………………………………...109 Chapter 5: Appendix……………………………………………………………….135 5 List of Abbreviations 5-OH-C 5-hydroxycytosine 5-OH-U 5-hydroxyuracil 8-oxoG 8-oxo-7,8-dihydroguanine A Adenine ALL Acute lymphoblastic leukaemia ALT Alternative lengthening of telomeres APC Adenomatous polyposis coli AP site Apurinic/Apyrimidinic site APE AP-endonuclease Bcl-2 B-cell lymphoma 2 BER Base excision repair B-ME 2-mercaptoethanol bp Base pair C Cytosine cALL common ALL cDNA Complementary DNA CEM-1-15 Human acute lymphoblastic leukaemia steroid resistant cell line CEM-7-14 Human acute lymphoblastic leukaemia steroid sensitive cell line Cg cytosine glycol CML Chronic myeloid leukaemia CNS Central nervous system CSR Class switch recombination Ct Threshold cycle. DEX Dexamethasone DHFR Dihydrofolate reductase DMEM Dulbecco's Modified Eagle's Medium 6 DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleoside triphosphates DOX Doxorubicin DSB Double-strand break dsDNA double-stranded DNA EDTA Ethylene diamine tetra acetic acid ETC Electron transport chain FapyA 4,6-diamino-5-formamidopyrimidine FapyG 2,6-diamino-4-hydroxy-5-formamidopyrimidine FBS Foetal bovine serum Fpg Formamidopyrimidine DNA glycosylase G Guanine G4 Guanine quadruplex Gapdh Glyceraldehyde-3-phosphate dehydrogenase GCs Glucocorticoids Gh Guanidinohydantoin GPXs Glutathione peroxidases GR Glucocorticoid receptor GSH Glutathione GSSG Glutathione disulphide GST P1 Glutathione S-transferase P1 H2O2 Hydrogen peroxide H2TH Helix 2 turn Helix HB Hepatoblastoma HepG2 Human hepatoblastoma cell line HIV-1 Human immunodeficiency virus type 1 hNEIL3 Human NEIL3 HOS Human osteogenic osteosarcoma cell line 7 hTERC Human telomerase RNA component hTERT Human telomerase reverse transcriptase lncRNAs Long non-coding RNAs MB Medulloblastoma MLL Mixed-lineage leukaemia MMR Mismatch repair Mmu NEIL3 Mus musculus NEIL3 nDNA Nuclear DNA NEIL nei-like NER Nucleotide excision repair NLS Nuclear localization signal Nth1 Endonuclease III homologue O2- Superoxide anion OGG1 8-oxoguanine DNA glycosylase OH. Hydroxyl free radical OS Osteosarcoma PBS Phosphate buffered saline PCNA Proliferating cell nuclear antigen PCR Polymerase chain reaction Ph chromosome Philadelphia chromosome PMS Phenazine methosulfate Pol DNA polymerase POT1 Protection of telomere protein 1 PRED Prednisolone Pro2 Proline amino acid at position 2 PRXs Peroxiredoxins qRT-PCR Quantitative real-time PCR RAR Replication-associated repair RE1 Repressor element 1 8 RNA Ribonucleic acid ROS Reactive oxygen species rRNA ribosomal RNA Saos-2 Human osteosarcoma methotrexate resistant cell line SHH Sonic Hedgehog