The Cytotoxic Effects of Malondialdehyde on Human Lung Fibroblast Cells Sally Ann Yates A thesis submitted in partial fulfilment of the requirements of Liverpool John Moores University for the degree of Doctor of Philosophy March 2015 Abstract ABSTRACT Malondialdehyde (MDA) is a mutagenic and carcinogenic product of lipid peroxidation which has also been found at elevated levels in smokers. MDA reacts with nucleic acid bases to form pyrimidopurinone DNA adducts, of which 3-(2-deoxy-β-D-erythro- pentofuranosyl)pyrimidol[1,2-α]purin-10(3H)-one (M1dG) is the most abundant and has been linked to smoking. Mutations in the TP53 tumour suppressor gene are associated with half of all cancers. This research applied a multidisciplinary approach to investigate the toxic effects of MDA on the human lung fibroblasts MRC5, which have an intact p53 response, and their SV40 transformed counterpart, MRC5 SV2, which have a sequestered p53 response. Both cell lines were treated with MDA (0-1000 µM) for 24 and 48 h and subjected to a variety of analyses to examine cell proliferation, cell viability, cellular and nuclear morphology, apoptosis, p53 protein expression, DNA topography and M1dG adduct detection. For the first time, mutation sequencing of the 5’ untranslated region (UTR) of the TP53 gene in response to MDA treatment was carried out. The main findings were that both cell lines showed reduced proliferation and viability with increasing concentrations of MDA, the cell surface and nuclear morphology were altered, and levels of apoptosis and p53 protein expression appeared to increase. A LC-MS-MS method for detection of M1dG adducts was developed and adducts were detected in CT-DNA treated with MDA in a dose-dependent manner. DNA appeared to become more fragmented with increasing MDA concentration, and the number of mutations in the 5’ UTR region of the TP53 gene also increased. The majority of mutations observed were insertions, compared to lung cancer mutation data where the majority were G to T transversions. This was unexpected, suggesting that tobacco smoke compounds have a different role in mutagenesis than endogenous lipid peroxidation. Thus, MDA has been found to have a clear effect on human lung fibroblasts at both the cellular and DNA level. i Acknowledgements ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisors, Dr Sharon Moore and Dr Mark Murphy, for their continued patience, support and guidance throughout the course of my project. I would also like to thank Dr Nicola Dempster for her assistance with LC-MS and LC-MS-MS, Campbell Woods and Phil Salmon for their help with HPLC, and Pete Hamlett for his help and guidance. I would like to thank Lieslot Hemeryck and Dr Lynn Vanhaecke of Ghent University, Belgium, for analysing a number of my samples by LC-MS-MS for independent verification. Finally, I would like to thank my husband, family and friends for their patience, encouragement and support throughout all of my years of study, in particular during my PhD. ii Table of Contents TABLE OF CONTENTS Abstract _______________________________________________________________ i Acknowledgements ____________________________________________________ ii Table of Contents ______________________________________________________ iii List of Figures ________________________________________________________ vii List of Schemes _______________________________________________________ xiv List of Tables _________________________________________________________ xvi List of Abbreviations __________________________________________________ xviii Chapter 1 _____________________________________________________________ 1 Introduction __________________________________________________________ 1 1.1. Cancer Incidence _____________________________________________________ 1 1.2. Development of Cancer ________________________________________________ 3 1.3. The TP53 Tumour Suppressor Gene ______________________________________ 5 1.4. Apoptosis __________________________________________________________ 10 1.5. Reactive Oxygen Species ______________________________________________ 11 1.6. Oxidative Stress _____________________________________________________ 13 1.7. Malondialdehyde ____________________________________________________ 14 1.8. Formation of Malondialdehyde ________________________________________ 16 1.9. Formation and Repair of the M1dG Adduct _______________________________ 19 iii Table of Contents 1.10. Analytical Methods Applied to Detection of Malondialdehyde and the M1dG Adduct __________________________________________________________________24 1.11. MRC5 and MRC5 SV2 Lung Fibroblast Cells _______________________________ 33 1.12. Imaging Techniques _________________________________________________ 35 1.13. Summary __________________________________________________________ 40 1.14. Experimental Design _________________________________________________ 41 1.15. Aims & Objectives ___________________________________________________ 45 Chapter 2 ____________________________________________________________ 46 Materials & Methods __________________________________________________ 46 2.1. Materials ___________________________________________________________ 46 2.2. Equipment _________________________________________________________ 49 2.3. Methods ___________________________________________________________ 50 Chapter 3 ____________________________________________________________ 81 Cellular Effects of Malondialdehyde ______________________________________ 81 3.1. Verification of Malondialdehyde Concentrations __________________________ 82 3.2. Effect of Foetal Bovine Serum and Malondialdehyde on Cell Growth and Viability__________________________________________________________________87 3.3. Cell Viability using a MTT Assay ________________________________________ 94 3.4. In vitro Imaging of Cells ______________________________________________ 101 3.5. Cell Surface Morphology by Atomic Force Microscopy _____________________ 106 3.6. Effects of MDA on Nuclear Morphology ________________________________ 110 iv Table of Contents 3.7. Apoptosis Detection using an Annexin V Fluorescent Assay ________________ 118 3.8. p53 Expression using Immunoflurescence _______________________________ 125 3.9. p53 Expression using Reverse Transcription Quantitative Polymerase Chain Reaction _________________________________________________________________ 129 3.10. Summary __________________________________________________________ 134 Chapter 4 ___________________________________________________________ 136 Adduct Analysis _____________________________________________________ 136 4.1. Synthesis of M1G Standard ___________________________________________ 136 4.2. High Performance Liquid Chromatography Method Development ___________ 140 4.3. Mass Spectrometry Method Development ______________________________ 150 4.4. Analysis of M1G Adducts in DNA _______________________________________ 163 4.5. Summary __________________________________________________________ 168 Chapter 5 ___________________________________________________________ 170 Effect of Malondialdehyde on DNA ______________________________________ 170 5.1. Imaging of DNA by Atomic Force Microscopy ____________________________ 170 5.2. Mutation Sequencing _______________________________________________ 176 5.3. Summary __________________________________________________________ 195 Chapter 6 ___________________________________________________________ 197 Conclusions & Future Work ____________________________________________ 197 6.1. Conclusions ________________________________________________________ 197 6.2. Future Work _______________________________________________________ 199 v Table of Contents References _________________________________________________________ 204 Appendix 1 _________________________________________________________ 235 Appendix 2 _________________________________________________________ 239 vi List of Figures LIST OF FIGURES Figure 1. The six acquired functional capabilities of cancer cells (derived from Hanahan and Weinberg, 2000).…………………………………………………………………………………………………….……4 Figure 2. p53 signalling pathways in response to DNA damage. (Adapted from SABiosciences.com (2015)). .......................................................................................... 6 Figure 3. p53 core domain. Image from the RCSB PDB (Wang, 2007) .................................... 7 Figure 4. TP53 isoforms: exons 1 – 11 and introns 6 and 9 are shown as boxes with promoters P1 (for p53 and Δ40p53 initiation of transcription) and P2 (for Δ 133p53 initiation of transcription) and alternative start codons (ATG) for translation start sites. No splicing of intron 2 occurs in Δ40p53 transcripts. TP53 α transcripts have normal splicing of exons with intron 9 fully spliced. TP53 β and γ transcripts have alternative splicing of intron 9. (Adapted from Wei et al (2012)). .................................................................................. 9 Figure 5. AFM principle: as the tip which is attached to the cantilever scans the sample surface, a laser beam is reflected off the back of the tip on to a photodiode detector which measures the deflection of the cantilever, allowing a 3-D image to be produced from the data (adapted from Billingsley et al. (2012)). ................................................. 38 Figure 6. AFM modes: A) tapping mode and B) contact mode, showing the tips’ movement over the sample surface (adapted from Santos and Castanho (2004)). .........................