i Investigating telomere dynamics using standard and AuNP-based assays and developing an LRP-based nanoparticle drug by Martin Bernert (375076) Thesis Submitted in fulfilment of the requirements for the degree Philosophiae Doctor in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, South Africa Supervisors: Prof Stefan FT Weiss and Dr. Eloise van der Merwe November 2023 ii Declaration I, Martin Bernert (375076), am a student registered for the degree of Doctor of Philosophy Science by Thesis (PhD) in the academic year 2023. I hereby declare the following: • I am aware that plagiarism (the use of someone else’s work without their permission and/or without acknowledging the original source) is wrong. • I confirm that the work submitted for assessment for the above degree is my own unaided work except where explicitly indicated otherwise. • I have not submitted this work before for any other degree or examination at this or any other University. • I have followed the required conventions in referencing the thoughts and ideas of others. • I understand that the University of the Witwatersrand may take disciplinary action against me if there is a belief that this is not my own unaided work or that I have failed to acknowledge the source of the ideas or words in my writing. Signed: 6th day of November 2023 iii Abstract Telomere dynamics, specifically telomerase activity have been implicated in age-related diseases, such as CVD, Alzheimer’s disease, and cancer. This makes the accurate detection of telomerase activity within cell cultures and tissue samples a necessity. Conventional techniques have many drawbacks, including their very high cost. Therefore, this research aimed to develop a gold nanoparticle (AuNP)-based assay to determine telomerase activity. In the assay the extracted telomerase leads to a colour change in the solution through the addition of telomeric repeats and subsequent elongation of the synthetic telomeres attached to the AuNPs. This colour change is detectable using spectrophotometric readings and represents telomerase activity. This assay would be useful as an alternative to expensive existing telomerase activity kits as large batches of AuNPs can be synthesised inexpensively. Telomerase activity was successfully detected in both HEK-293 and WHCO-5 cells using this novel technique, although the sensitivity of the AuNP-based telomerase activity assay is currently lower than a commercially available qPCR-based telomerases activity kit. In addition, telomerase activity is directly affected by the LRP protein, a highly conserved non-integrin transmembrane receptor, which has been shown to have therapeutic effects in ageing, Alzheimer’s disease, Parkinson’s disease, diabetes, and cardiovascular disease models. Recently it has been found that overexpression of LRP::FLAG, by plasmid transfection, leads to a significant increase in telomerase activity in cell culture models. This may indicate that upregulation of LRP can be used to treat various age-related diseases, however, transfection is not a viable treatment strategy and therefore, a protein-based drug was created. For a protein-based drug, a suitable delivery system needed to be developed and nano-capsules, such as those synthesised using Poly(lactic-co-glycolic acid) (PLGA), are able to contain the therapeutic protein. The molecules contained within the nanoparticles also gain the benefit of having increased stability compared to unprotected molecules and the capsules have the capacity for surface modifications for targeted therapy. These polymer- based nanoparticles are also biodegradable and biocompatible, making them a safe delivery agent. Thus, this research further aimed to develop a PLGA-based LRP drug delivery system for the 37 kDa Laminin receptor protein. Both synthesis of the nanoparticles and encapsulation of the LRP protein were successfully optimised and the completed drug was tested in a cell culture model, where treatment increased cell viability and telomerase activity in HEK-293 cells. Therefore, this LRP drug delivery system has great potential to assist in the translation of our in vitro studies into an in vivo context. Due to the wide range of applications elevating LRP levels has in the treatment of different disorders, this could represent a safer alternative to plasmid transfection treatment and could potentially be used for the treatment of age-related diseases, through its ability to increase telomerase activity. iv Acknowledgements Firstly, I would like to thank Dr. Eloise van der Merwe for being not just an incredible supervisor but an amazing friend. Your excitement for your work is infectious and this has kept me motivated through rough times. You have pushed and encouraged me to take on new and challenging scientific techniques and I cannot thank you enough for this. Throughout my entire postgraduate journey, you have been a constant source of knowledge and support, without which I would never have come as far as I have. I would also like to thank my parents, Albert, and Monika Kopp, for everything they have done to support me throughout my PhD. Your genuine interest in everything I do has been an incredible motivator and I don’t have the words to adequately convey the love and respect I have for you both. Thank you for being the best parents I could have wished for. And Albert, I am happy to finally have a satisfactory answer to your question: yes, it is finally done! I would also like to thank my girlfriend, Claire Tinderholm. Thank you for your infinite patience in dealing with my ups and downs, your presence alone has made my life so much better. I could not have done this without your help and support, and I cannot thank you enough. Thank you to my friends, Nick, Gen, Bacci, Bradley, Dave and Andi. You guys are awesome, thank you for the games, intellectual and not so intellectual conversations as well as looking out for me. You guys kept me motivated. I would like to give a very special thank you to Dr. Tyrone Otgaar, Dr. Monique Bignoux and Dr. Gavin Morris. Tyrone, you have been the rock that kept our lab together and I am grateful to have you as one of my friends. Your passion for science is evident in every conversation we have v had, and I have learned a so much from you. Monique, you bring a vibe to the lab I can’t fully explain, you can make the heaviest subject fun and see a lack of a smile on your colleagues faces as a personal challenge. Thank you for all your help and support over the years. Gavin, you have been a fantastic friend and roommate to me, your assistance during my PhD has been invaluable and your insights profound. You are one of the smartest people I know (and I know a lot of smart people), thank you for everything you have done for me. I thoroughly enjoyed our lengthy troubleshooting sessions together and I wish you three all the best for the future. Thank you to my colleagues of Weiss lab and the Cell Biology and Signalling Research lab, you all have made an impact on me. It has been a privilege to see so many of you become the amazing scientists you are today. I would like to specifically acknowledge Chandni Madhav and Sichumiso Gqeba for your assistance with the protein work. It was tough stuff. Finally, I would like to thank my late supervisors Dr. Boitelo Letsolo and Prof. Stefan Weiss. Although you are no longer with us, your fierce intelligences and personalities have made an impact on the world. Dr. Letsolo was my first supervisor and the reason I fell in love with molecular biology. You strove to not only make me a better scientist but also a better person. Similarly, Prof. Weiss was not just a supervisor, he was a force of nature and everyone who knew him was better off for it. He encouraged me to challenge and to better myself in all things. I will always remember being hurriedly pulled out of the culture lab just to play a round of darts with you. You both are dearly missed. Financial aid: Wits Postgraduate Merit Award (PGMA) (2015-2016) National Research Foundation (NRF) – Free Standing (2016) Technology Innovation Agency (TIA) Seed Fund (2017-2023) University of the Witwatersrand seed fund (2021) vi Research outputs Publications: • Bignoux MJ, Otgaar TC, Bernert M, Weiss SFT and Ferreira E (2022) siRNA- mediated downregulation of LRP/LR inhibits multiple cancer hallmarks in lung cancer cells. FEBS One Bio (under review). • Vania L, Morris G, Otgaar TC, Bignoux MJ, Bernert M, Burns J, Gabathuse A, Singh E, Ferreira E and Weiss SFT (2019) Patented therapeutic approaches targeting LRP/LR for cancer treatment. Expert Opinion on Therapeutic Patents. 29(12):987-1009 • Otgaar TC, Ferreira E, Malindisa S, Bernert M, Letsolo BT and Weiss SFT (2017) 37 kDa LRP::FLAG enhances telomerase activity and reduces senescent markers in vitro. Oncotarget. 8(49):86646-86656. doi:10.18632/oncotarget.21278. • Naidoo K, Malindisa ST, Otgaar TC, Bernert M, Da Costa Dias B, Ferreira E, Reusch U, Knackmuss S, Little M, Weiss SFT and Letsolo BT (2015) Knock-down of the 37kDa/67kDa laminin receptor LRP/LR impedes telomerase activity. Plos One. 10(11) Patents: • South Africa patent application WO/2019/116334 is in the name of the University of the Witwatersrand entitled: “A Nanoparticle-Based Telomerase Assay.” Inventors: Stefan Weiss, Boitelo Teresa Letsolo, Martin Bernert. vii • South Africa patent application WO/2020/008442A3 is in the name of the University of the Witwatersrand entitled: “Biopharmaceutical agents for use in reducing lipid content in cells”. Inventors: Stefan Weiss, Eloise van der Merwe, Martin Bernert and Tyrone Otgaar. Conference outputs: • Bernert M., Letsolo B. T. Metformin Reduces Telomerase Activity in Oesophageal Carcinoma Cells Expressing Wild-Type p53. Poster Presentation. Molecular Biosciences Research Thrust (MBRT) Research Day, Johannesburg, South Africa. 2014 • Bernert M., Otgaar T. C., Baichan P., Malindisa S.T., Weiss S.F.T, Veale R. B., Moeno S. and Letsolo B.T. Investigating Telomere Dynamics in Oesophageal Squamous Carcinoma Cells using Standard and Nanoparticle-based Assays. Poster Presentation. Molecular Biosciences Research Thrust (MBRT) Research Day. Johannesburg, South Africa. 2016. • Bernert M., Kondiah K., Veale R.B., Moeno S., Letsolo B.T., and Weiss S.F.T. Investigating Telomere Dynamics in Oesophageal Squamous Carcinoma Cells using Standard and Nanoparticle-based Assays. Poster presentation. Molecular Biosciences Research Thrust (MBRT) Research Day, Johannesburg, South Africa. 2017 • Bernert M., Kondiah K., Veale R.B., Moeno S., Letsolo B.T., and Weiss S.F.T. Investigating Telomere Dynamics in Oesophageal Squamous Carcinoma Cells using Standard and Nanoparticle-based Assays. Oral presentation. South African Society for Biochemistry and Molecular Biology (SASBMB). Potchefstroom, South Africa. 2018. viii • Bernert M., Kondiah K., Veale R.B., Moeno S., Letsolo B.T., and Weiss S.F.T. Investigating Telomere Dynamics in Oesophageal Squamous Carcinoma Cells using Standard and Nanoparticle-based Assays. Poster presentation. South African Society for Biochemistry and Molecular Biology (SASBMB). Potchefstroom, South Africa. 2018. • Bernert M., Morris G., Veale R., Ferreira E., and Weiss S.F.T. Developing Nanoparticle-based Systems for the Detection of Telomerase Activity as well as Drug Delivery. Poster presentation. Molecular Biosciences Research Thrust (MBRT) Research Day, Johannesburg, South Africa. 2019 • Ferreira E., Otgaar T.C., Bernert M., Morris G., Baichan P., Letsolo B.T. and Weiss S.F.T. In vivo investigation of the effect of LRP::FLAG overexpression on the ageing process and telomere dynamics in mice. Oral presentation, OLC International Biotechnology Conference, Chicago, USA, 23-24 September 2019 ix Table of Contents Declaration .......................................................................................................................................ii Abstract ........................................................................................................................................... iii Acknowledgements ......................................................................................................................... iv Research outputs ............................................................................................................................ vi Table of Contents ............................................................................................................................ ix List of Figures ................................................................................................................................ xiii List of Tables .................................................................................................................................. xv List of Abbreviations ..................................................................................................................... xvi 1. Introduction and Literature Review ........................................................................................ 1 1.1. Telomeres ......................................................................................................................... 1 1.1.1. Function .................................................................................................................... 1 1.1.2. Structure ................................................................................................................... 3 1.1.3. Telomerase................................................................................................................ 5 1.1.4. Extratelomeric functions of hTERT ........................................................................... 8 1.2. Telomeres in diseases ...................................................................................................... 8 1.2.1. Telomeres and cancer ............................................................................................... 8 1.2.2. The role of telomeres in age related disorders ...................................................... 13 1.2.3. The role of telomeres in neurodegenerative disorders .......................................... 14 1.3. Telomerase modulators ................................................................................................. 15 1.3.1. Metformin inhibits telomerase activity .................................................................. 15 1.3.1. The MST-312 telomerase inhibitor ......................................................................... 17 1.3.2. The TA-65 telomerase enhancer............................................................................. 17 x 1.4. 37 kDa laminin receptor precursor/67 kDa high affinity laminin receptor ................... 18 1.4.1. Structure and function ............................................................................................ 18 1.4.2. LRP/LR and telomerase ........................................................................................... 19 1.4.3. The role of LRP/LR in cancer ................................................................................... 20 1.4.4. The role of LRP/LR in neurodegenerative disease .................................................. 21 1.5. Applications of nanoparticles in drug delivery and research......................................... 22 1.5.1. PLGA nano-capsules as drug delivery agents ......................................................... 22 1.5.2. Nanoparticles in telomerase activity assays ........................................................... 23 1.6. Research rationale.......................................................................................................... 26 2. Aims and Objectives .............................................................................................................. 28 2.1. Aim 1: Optimise the gold nanoparticle telomerase activity assay and improve the stability of the functionalised AuNPs by redesigning separate linker and extension strands. 28 2.2. Aim 2: Create an LRP based nanoparticle drug. ............................................................. 28 3. Materials and Methods ......................................................................................................... 29 3.1. Cell culture ..................................................................................................................... 29 3.1.1. Cell culture protocol ............................................................................................... 29 3.1.2. Passaging of cells .................................................................................................... 29 3.1.3. Cryo-preservation of cells ....................................................................................... 30 3.1.4. Cell quantification ................................................................................................... 30 3.1.5. Metformin and MST-312 Treatments ..................................................................... 31 3.1.6. MTT cell viability assay ............................................................................................ 32 3.1.7. DNA quantification - agarose gel electrophoresis .................................................. 32 3.1.8. Telomerase activity ................................................................................................. 33 3.2. Section 1: Gold nanoparticle telomerase activity assay ................................................ 35 xi 3.2.1. AuNP synthesis ........................................................................................................ 36 3.2.2. AuNP DNA functionalisation ................................................................................... 36 3.2.3. AuNP based telomerase activity assay ................................................................... 38 3.2.4. Electron microscopy (SEM and TEM) and spectrophotometry .............................. 40 3.3. Section 2: LRP-based PLGA nanoparticle drug design ................................................... 41 3.3.1. Nanoparticle drug design ........................................................................................ 41 3.3.2. BCA/ micro-BCA assay ............................................................................................. 43 3.3.3. Western blot ........................................................................................................... 43 3.3.4. Telomerase activity ................................................................................................. 45 3.4. Data analysis and statistical evaluation ......................................................................... 45 4. Results ................................................................................................................................... 46 4.1. Section 1: AuNP-based telomerase activity assay ......................................................... 46 4.1.1. Metallic nanoparticle synthesis .............................................................................. 47 4.1.2. AuNP characterisation ............................................................................................ 49 4.1.3. Thiol-DNA Functionalisation of gold nanoparticles ................................................ 53 4.1.4. DNA functionalisation - pH dependent method ..................................................... 57 4.1.5. AuNP-based telomerase activity assay ................................................................... 60 4.1. Section 2: Encapsulated-LRP PLGA nano-capsules ........................................................ 65 4.1.1. Optimisation of PLGA nano-capsule synthesis ....................................................... 66 4.1.2. The effects of empty PLGA nano-capsules on cell viability .................................... 77 4.1.3. Protein encapsulation using PLGA nano-capsules .................................................. 79 4.1.4. LRP protein encapsulation ...................................................................................... 83 4.1.5. Effect of encapsulated LRP treatments on total LRP levels, cell viability and telomerase activity ............................................................................................................... 85 xii 5. Discussion.............................................................................................................................. 90 5.1. Section 1: AuNP-based telomerase activity assay ......................................................... 90 5.1.1. Synthesis and characterisation ............................................................................... 91 5.1.2. DNA functionalisation ............................................................................................. 92 5.1.3. AuNP-based telomerase activity assay ................................................................... 94 5.1.4. AuNP section conclusion ......................................................................................... 97 5.1.5. Future research for the AuNP-based telomerase activity assay ............................ 98 5.2. Section 2: LRP-encapsulated PLGA nano-capsules ........................................................ 99 5.2.1. Optimisation of synthesis ....................................................................................... 99 5.2.2. Purification of PLGA nano-capsules ...................................................................... 101 5.2.3. Protein encapsulation trials .................................................................................. 104 5.2.4. LRP-PLGA nano-capsule treatments ..................................................................... 106 5.2.5. PLGA section conclusion ....................................................................................... 109 5.2.6. Future research for the LRP-encapsulated PLGA nano-capsules ......................... 109 5.3. Conclusion .................................................................................................................... 110 6. References .......................................................................................................................... 112 7. Supplementary results ........................................................................................................ 127 7.1. Metformin treatment reduces telomerase activity on oesophageal cancer cells: ...... 127 7.2. LRP protein synthesis ................................................................................................... 128 8. Supplementary information................................................................................................ 137 8.1. List of reagents and materials: ..................................................................................... 137 8.2. List of equipment: ........................................................................................................ 138 8.3. List of software: ............................................................................................................ 138 xiii List of Figures Figure 1.1: Schematic representation of the "end-replication" problem ...................................... 2 Figure 1.2: Focused view of the shelterin complex ........................................................................ 4 Figure 1.3: Telomere secondary structures and associated stabilising proteins ........................... 5 Figure 1.4: Schematic representation of the telomerase enzyme ................................................. 6 Figure 1.5: The relationship between telomere shortening and cancer generation ................... 10 Figure 1.6: Flow diagram of cellular crisis ..................................................................................... 11 Figure 1.7: Schematic representation of the LRP/LR transmembrane receptor, binding various biomolecules ................................................................................................................................. 19 Figure 1.8: Principles of a AuNP-based telomerase activity assay ............................................... 26 Figure 3.1: Predicted results when using the improved AuNP-based telomerase activity assay 40 Figure 4.1: Optimisation of AuNP synthesis parameters .............................................................. 48 Figure 4.2: Spectrophotometric analysis of AuNP synthesis reactions ........................................ 50 Figure 4.3: A610/520 ratio confirms the consistency of the AuNP synthesis reactions ................... 51 Figure 4.4: AuNP size distribution determined by TEM and ImageJ analysis ............................... 52 Figure 4.5: Schematic representation of the first iteration of the AuNP-based telomerase activity assay .............................................................................................................................................. 54 Figure 4.6: Transmission electron microscopy analysis of 5’ extension strand functionalised AuNPs ............................................................................................................................................ 55 Figure 4.7: Schematic diagram of the Improved AuNP telomerase activity assay ....................... 56 Figure 4.8: Annealing of telomerase extension strand and thiolated linker DNA ........................ 57 Figure 4.9: Spectrophotometric comparison between thiol-DNA functionalised and non- functionalised AuNPs .................................................................................................................... 58 Figure 4.10: Comparison of DNA-functionalised and non-functionalised AuNP, using the A610/520 ratio ............................................................................................................................................... 59 Figure 4.11: Transmission electron micrograph of DNA functionalized AuNPs ........................... 60 Figure 4.12: Successful detection of telomerase activity using the improved AuNP based telomerase activity assay .............................................................................................................. 61 xiv Figure 4.13: Relative telomerase activity of HEK-293 and WHCO-5 cells after MST-312 telomerase inhibitor and metformin treatment (TRAPeze® RT Telomerase Detection Kit) ........................... 62 Figure 4.14: Relative telomerase activity of HEK-293 and WHCO-5 cells after MST-312 telomerase inhibitor and metformin treatment (AuNP-based telomerase activity assay) ............................. 64 Figure 4.15: Light microscope and TEM images of PLGA nano-capsules ..................................... 67 Figure 4.16: Initial PLGA nano-capsule synthesis ......................................................................... 68 Figure 4.17: The effect of initial PLGA (in solvent) concentration on nano-capsule polydispersity ....................................................................................................................................................... 70 Figure 4.18: PLGA nano-capsules purification by centrifugation at 8000 xg ................................ 71 Figure 4.19: PLGA nano-capsules purification by centrifugation at 10 000 xg ............................. 72 Figure 4.20: PLGA nano-capsules purification by centrifugation at 12 000 xg ............................. 73 Figure 4.21: PLGA nano-capsules purification by centrifugation at 14 000 xg ............................. 74 Figure 4.22: PLGA nano-capsules purification by 0.45 µl filtration and centrifugation at 500 xg 75 Figure 4.23: Evaluation of the size distribution of PLGA nano-capsules ...................................... 77 Figure 4.24: HEK-293 cell viability after PLGA nano-capsule treatment ...................................... 78 Figure 4.25: Scanning electron micrograph of protein encapsulated PLGA nanoparticles .......... 79 Figure 4.26: BCA assay results of encapsulated-BSA PLGA nanoparticles ................................... 80 Figure 4.27: Micro-BCA assay results of encapsulated-GFP PLGA nano-capsules ....................... 82 Figure 4.28: Micro-BCA assay results of empty and encapsulated-LRP PLGA nanoparticles ....... 84 Figure 4.29: Western blot analysis of encapsulated-LRP PLGA nanoparticle treatments on HEK- 293 cells ........................................................................................................................................ 86 Figure 4.30: Improved cell viability after LRP-nanoparticle treatment in vitro ........................... 87 Figure 4.31: LRP-nanoparticle treatment elevates telomerase activity in vitro .......................... 88 Figure 7.1: Metformin reduces telomerase activity in WHCO-1 cells ........................................ 127 Figure 7.2: LRP protein sequence homology .............................................................................. 129 Figure 7.3: Initial expression of LRP at 37 °C using varying IPTG concentrations ...................... 130 Figure 7.4: Initial IMAC purification of LRP ................................................................................. 131 Figure 7.5: Coomassie blue stained SDS-PAGE gel of the purified LRP ...................................... 132 Figure 7.6: Expression of LRP at 20 °C using varying IPTG concentrations................................. 133 xv Figure 7.7: Purification of LRP using IMAC ................................................................................. 133 Figure 7.8: Improved IMAC purification of LRP .......................................................................... 134 Figure 7.9: Coomassie blue stained SDS-PAGE gel of the purified LRP ...................................... 135 Figure 7.10: Confirmation of purified LRP via standard curve extrapolation ............................. 136 List of Tables Table 3.1: Telomerase activity primers ......................................................................................... 35 Table 3.2: Oligo sequences for the improved AuNP-based telomerase activity assay. ............... 38 Table 4.1: DLS results of PLGA nano-capsules .............................................................................. 76 xvi List of Abbreviations Abbreviation Meaning µl microliter µM micromolar µm micrometre ALT alternative lengthening of telomeres AMP adenosine monophosphate AMPK AMP-activated protein kinase AMPK adenosine monophosphate activated protein kinase AuNP gold nanoparticle Aβ amyloid-beta BCA bicinchoninic acid BSA bovine serum albumen CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate CVD cardiovascular disease DCM dichloromethane d-loop displacement loop DLS dynamic light scattering DMEM Dulbecco's modified eagle's medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP deoxyribonucleotide dsDNA double stranded DNA EA ethyl acetate EDTA ethylene diaminetetraacetic acid EGCG epigallocatechin gallate EGTA ethylene glycol-bis(β-aminoethyl ether) ELISA enzyme-linked immunosorbent assay FBS foetal bovine serum GFP green fluorescent protein hTERC human telomerase RNA component hTERT human telomerase reverse transcriptase component IC50 50% inhibitory concentration IMAC immobilized metal chelate affinity chromatography IPTG Isopropyl β-D-1-thiogalactopyranoside kbp kilo base pairs LDL low-density lipoprotein LKB1 liver kinase B1 LRP/LR 37 kDa laminin receptor precursor/67 kDa high affinity laminin receptor M molar mg milligram min minutes xvii ml millilitre mm millimetre mM millimolar mRNA messenger ribonucleic acid MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ng nanogram nm nanometer nM nano molar oxLDL oxidised low-density lipoprotein PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction PDI polydispersity index PEG polyethylene glycol PLGA poly(lactic-co-glycolic acid) POT1 protection of telomeres 1 pRb phosphorylated Retinoblastoma protein PrPc scrapie prion protein PrPSc scrapie prion protein PVA polyvinyl alcohol qPCR quantitative polymerase chain reaction RAP1 human repressor activator protein RIPA buffer radioimmunoprecipitation assay buffer RNA ribonucleic acid RNase ribonuclease ROS reactive oxygen species SDS sodium dodecyl sulphate SEM scanning electron microscopy SNPs single nucleotide polymorphisms ss-DNA single stranded DNA STELA Single telomere length analysis TBE tris/borate/EDTA TE tris/EDTA TEM transmission electron microscopy TIN2 TRF1-interacting nuclear protein 2 t-loop telomere loop TPP1 adrenocortical dysplasia protein TRAP telomeric repeat amplification protocol TRF telomeric repeat-binding factor 2 TRF1 telomeric repeat-binding factor 1 V volts VNTR variable number of tandem repeats Xg times gravity 1 1. Introduction and Literature Review 1.1. Telomeres 1.1.1. Function Telomeres are non-coding repetitive double stranded TTAGGG DNA repeats at the ends of chromosomes and are between 10-15 kbp in length in humans (Fleming & Burrows, 2013). These telomeres help stabilise and protect the ends of chromosomes. They carry out this protective function by preventing the erosion of important coding DNA by the “end-replication” problem (Levy et al., 1992). This loss of coding DNA occurs because of the incomplete synthesis of double stranded DNA, which is a characteristic of the mode of action of RNA dependant DNA polymerase (Figure 1.1). The polymerase utilises an RNA primer in order to synthesise a new DNA strand. Due to the degradation of this RNA primer, a single stranded overhang remains after lagging strand synthesis (Levy et al., 1992). This overhang is then removed, which results in the loss of small fragments of DNA after each synthesis reaction (reviewed in Denchi, 2009) and this, therefore, leads to the systematic erosion of the ends of chromosomes after cell division (Blackburn, 1991). 2 Figure 1.1: Schematic representation of the "end-replication" problem The DNA synthesis reaction, performed by DNA polymerase, involves both the leading strand (green) and the lagging strand (blue) of the replication fork are shown with an attached RNA primer. As this RNA primer is degraded, the newly synthesised double stranded DNA is missing complimentary bases on the 5ˊend, leading to 3ˊ overhangs in the template strand. This overhang is eventually degraded resulting in the shortening of the DNA strand (Klapper et al., 2001; Levy et al., 1992). Telomeres act as a “buffer” zone to this erosion, however, if they are not continually maintained after each cell division, it can lead to the degradation of coding DNA, leading to a breakdown of normal functions if the cell does not enter a senescent state (where cells are metabolically active, but no longer replicate) (Shay & Wright, 2005). Telomerase, a multi-subunit enzyme, maintains and elongates the telomeres. However, it has been shown to be nearly undetectable in most somatic cell lines (Kim et al., 1994). This is significant, as it means that many mammalian cells are therefore susceptible to genetic ageing and undergoing senescence (Shay & Wright, 2005). In 3 contrast, germ-line cells in addition to other highly proliferating cell types, such as intestinal and oesophageal cells, have been shown to have high telomerase activity. This is most likely due to these cell types requiring to undergo constant cell division (Kim et al., 1994; Wright et al., 1996). If the telomeres are not maintained, become short and a functional DNA repair mechanism, it could lead to replicative senescence or even the induction of apoptosis (programmed cell death) (Shay & Wright, 2005). Normally, somatic cells are only capable of undergoing a limited number of cell divisions because of the shortening of telomeres. This is known as the “Hayflick limit” (Hayflick & Moorhead, 1961). When approaching this limit, it can lead to the disruption of normal tissue function, which has been theorised to contribute to the ageing process (Carneiro et al., 2016; Sahin & Depinho, 2010). This is further supported by the fact that older individuals have shorter telomeres leading to the telomere theory of ageing. 1.1.2. Structure The telomeric repeats, in conjunction with telomere stabilising proteins such as the shelterin complex (Figure 1.2), help stabilise the ends of chromosomes by maintaining telomere length and capping the ends of telomeres (Bailey & Murnane, 2006). The shelterin complex consists of multiple telomere-associated proteins namely, telomeric repeat-binding factor 1 and 2 (TRF1 and 2) and protection of telomeres 1 (POT1), which bind directly to the telomeric DNA, thereafter, allowing TRF1-interacting nuclear protein 2 (TIN2) to bind to TRF1 and TRF2 (Bailey & Murnane, 2006). Human repressor activator protein (RAP1) binds to TRF2, and adrenocortical dysplasia protein (TPP1) helps POT1 associate with the telomere. These proteins are also involved in the regulation of telomere length (Bailey & Murnane, 2006). The telomeres form secondary structures called the telomere loop (t-loop) and displacement loop (d-loop) (Figure 1.3). The t- and d-loops are formed when the telomere folds back on itself and integrates the 3’ overhang back into the telomere (Bailey & Murnane, 2006; Greider, 1999). The shelterin complex in conjunction with the t- and d-loops prevent the degradation of the telomeres and chromosome 4 end-fusions (Bailey & Murnane, 2006; Greider, 1999; Ohno et al., 2016). Chromosome end- fusions are the result of destabilised/unprotected telomeres, where two chromosomes are fused at their telomeric ends, possibly resulting in the disruption of mitosis and even cancer formation (Ohno et al., 2016). Figure 1.2: Focused view of the shelterin complex The major components of the “shelterin” complex are: TRF1/2, TIN2, RAP1, TPP1, and POT1. Here TRF1/2 allow for the binding of the complex to the double stranded DNA and POT1 to single stranded DNA whereas the other components help stabilise the complex (Bailey & Murnane, 2006; Raffa et al., 2013). 5 Figure 1.3: Telomere secondary structures and associated stabilising proteins This image shows the formation of the shelterin complex and depicts its stabilisation role in conjunction with the telomere loop (t-loop) and displacement loop (d-loop). The t-loop forms as the 3’ overhang folds in on itself (red DNA strand). The shelterin complex is shown with its major components; TRF1 (blue) and TRF2 (dark green) as stabilising the t-loop. The d-loop is formed as a result and is stabilised by RAP1 and POT1 (Bailey & Murnane, 2006; Greider, 1999; Kovacic et al., 2011). 1.1.3. Telomerase The telomerase enzyme is a ribonucleoprotein consisting of multiple subunits, two of which are essential. These subunits perform the main function of the enzyme, by maintaining telomere length. The human telomerase RNA component (hTERC) functions as an RNA template for the human telomerase reverse transcriptase component (hTERT), which adds the telomeric repeats to the ends of telomeres (Figure 1.4) (Carlin et al., 1997). It has been found that overexpression of hTERT in non-immortalised cell lines leads to cell immortalisation, whereas the overexpression of hTERC did not, this makes hTERT the limiting factor for enzyme activity (Sheng et al., 2013; Wick et al., 1999). In fact, it has even been shown that the increase of hTERT expression at any 6 stage of the cell cycle results in the immortalisation of the cell line (Wick et al., 1999). This is further supported with the fact that hTERT mRNA levels are directly proportional to telomerase activity. This correlates to the high number of cancers (90%) that have elevated telomerase activity, as they also exhibit increased hTERT mRNA levels (Kirkpatrick et al., 2003). Figure 1.4: Schematic representation of the telomerase enzyme The telomerase complex is made up of two major components: the telomerase reverse transcriptase component (TERT) and the telomerase RNA component/ template (TERC). The enzyme is stabilised by the presence of the proteins: dyskerin, GAR, NOP10 and NHP2. Telomerase maintains the telomeres by using TERC (blue) as a template for TERT (orange) to add additional telomeric repeats (TTAGGG) to the ends of chromosomes in a 3’ to 5’ direction, leaving a 3’ overhang (Calado & Young, 2009). 7 Since hTERT is the limiting factor of telomerase, this suggests that influences on hTERT mRNA expression could greatly affect telomerase activity. There are multiple factors such as the variation of the hTERT minisatellite (MNS16A) in addition to mutations of the hTERT promoter sequence, which have both been associated with hTERT expression (Wang et al., 2003). Additionally, specific single nucleotide polymorphisms (SNPs) that can be found in the hTERT promoter affect hTERT expression. In fact, an increased risk to lymphoblastic leukaemia and increased hTERT mRNA expression has been linked to the SNP rs2735940 in the hTERT promoter region (Sheng et al., 2013). Similarly, a genetic change in the hTERT minisatellite (MNS16A), which is a polymorphic tandem repeat downstream of the hTERT gene, has been linked to hTERT expression. It is thought that the antisense MNS16A gene is involved in the silencing of hTERT mRNA. This means that the shorter variant of the minisatellite has an increased ability to silence hTERT mRNA. The larger variants, on the other hand, would lead to decreased silencing due to a decrease in antisense transcription, and therefore cause an increase in hTERT expression (Wang et al., 2003). Further supporting this; the short allele of the variable number of tandem repeats (VNTR) of the MNS16A minisatellite (VNTR-274) has been found to downregulate hTERT mRNA expression (Hofer et al., 2013). This may lead to apoptosis or even senescence. VNTR-274 has even had a suggested protective role against prostate cancer (Hofer et al., 2013). In previous research, it was also found that cell lines containing the larger minisatellite variant had correspondingly high telomerase activity (Bernert et al., (unpublished); (Wang et al., 2003). Comparably, the proto-oncogene, c-myc, has been shown to increase hTERT expression. C-myc directly targets the hTERT gene, leading to increased telomerase activity due to increased hTERT expression (Greenberg et al., 1999; Tang et al., 2016). Even the p53 transcription factor has been known to target the hTERT gene, where it is thought to directly bind the hTERT gene through the SP1 binding site. This is said to regulate hTERT expression in normal, healthy cells by decreasing hTERT transcription (Kanaya et al., 2000). This, however, means that if there should be a loss of function mutation in p53, that it could lead to an increase in cell proliferation. 8 1.1.4. Extratelomeric functions of hTERT hTERT has been found to play a role not only in telomere maintenance and cell proliferation but has been implicated in a variety of extra-telomeric functions. During times of oxidative stress, hTERT is known to relocate to the mitochondria from the nucleus via a mitochondrial leader sequence present in the enzyme in order to improve mitochondrial function. This targeting sequence facilitates the shuttling of the enzyme into the mitochondria (Haendeler et al., 2009). The mitochondrial hTERT has been shown to improve mitochondrial function in times of oxidative stress, suggesting a potential protective role (Saretzki, 2009). Without this form of protection, it could lead to mitochondrial dysfunction, which can further cause the release of reactive oxygen species (ROS). Since ROS damage the genetic material of the cell (via oxidative damage), increased hTERT may protect mitochondrial DNA from damage by binding to it and preventing contact with ROS (Haendeler et al., 2009). Addditionally, hTERT has been implicated in DNA damage response, as it helps recruit DNA repair proteins (Saretzki, 2014). 1.2. Telomeres in diseases 1.2.1. Telomeres and cancer Cancer is one of the world’s leading causes of death and is characterised by abnormal cell proliferation (Jemal et al., 2011). Over 10 million deaths, from over 19.3 million cases, were reported in 2020 (GLOBOCAN 2020). Telomeres are vital for continued cell proliferation, and therefore have an important role in cancer genetics. Since continued cell division leads to telomere shortening, DNA repair pathways such as the p53/p21 pathway can be triggered, causing cell cycle arrest (senescence) and even apoptosis (Figure 1.5) (Shay & Wright, 2005). This occurs when the telomeres become critically short, or DNA breaks are detected by gamma H2AX. It then aids in the formation of DNA repair foci which in turn promotes the p53/p21 DNA damage response. Here the damage is either repaired or the cells are forced to undergo senescence or 9 apoptosis (Fragkos et al., 2009). Consequently, senescence acts as a tumour suppressor mechanism, which needs to be circumvented by cells in order for them to become malignant (Shay & Wright, 2005). In fact, our previous research has shown that p53 plays a significant role in decreasing telomerase activity. After metformin treatment of oesophageal cancer cells, it was thought that the AMPK pathway was activated, which in turn activated p53, and supressed hTERT promoter activity by repressing hTERT expression through the binding of the hTERT promoter SP1 binding sites (Bernert et al., (unpublished); Kanaya et al., 2000). The binding activity of p53 is activated through the ATM-dependent phosphorylation of serine 15 due to telomere loss. It is important to note, however, that this is not necessarily the only reason for this activation, age- related oxidative damage can also cause this phosphorylation (Kim et al., 2014). This suggests that mutations in the p53 tumour suppressor could directly increase telomerase activity and drive cancer cell immortality. Taking this into consideration, DNA damage response pathways and telomere dynamics can play a vital role in the progression of diseases such as cancer, due to its characteristic uncontrolled cell proliferation (Figure 1.6). This means that if cells lack sufficient DNA damage responses, such as in the case of mutated p53 or phosphorylated retinoblastoma protein (pRb), the cells could bypass senescence and become genetically unstable. This second checkpoint is known as “cellular crisis” (Figure 1.6 and Figure 1.5). “Cellular crisis” is characterised by critically short telomeres and chromosome instability, which can lead to telomere fusions (Capper et al., 2007). In order for cancer cells to continue proliferating at this stage, telomeres must either be maintained by the upregulation of telomerase (and therefore increased telomerase activity), or through alternative lengthening of telomeres (ALT) (Cesare & Reddel, 2010). ALT is recombination based and is utilised by approximately 10% of cancers, whereas telomerase upregulation can be found in up to 90% of cancers. ALT is utilised by cancers to avoid being recognised as DNA damage/ breaks, which could lead to chromosome end-fusions and further genomic instability. It uses homologous recombination (where chromosomes exchange telomeric material between one another) to maintain telomeres. This rare form of telomere maintenance is able to evade senescence and lead to cell immortalisation (Cesare & Reddel, 2010). This makes targeting telomeres and telomerase activity an interesting prospect for anticancer treatment. 10 Figure 1.5: The relationship between telomere shortening and cancer generation As the telomeres shorten after successive cell divisions, cells can either enter a senescent state or, by bypassing this, enter cellular crisis. At this point, if DNA damage response mechanisms are still active, the cell will undergo apoptosis. However, cell immortalisation may occur if these mechanisms are suppressed, potentially leading to cancer. 11 Figure 1.6: Flow diagram of cellular crisis This diagram shows the relationship of telomere shortening in the presence and absence of functional DNA repair mechanisms (phosphorylated retinoblastoma protein (pRb) and p53). In the presence of functional mechanisms, the cell will undergo apoptosis or replicative senescence, whereas without these mechanisms, telomere shortening can progress to cellular crisis. This can subsequently lead to genomic instability and even cancer. 1.2.1.1. Oesophageal cancer A cancer that has previously been shown to have high levels of telomerase is oesophageal cancer. Oesophageal cancer caused over 540,000 deaths, making it the 6th most deadly cancer in 2020 (GLOBOCAN 2020). Southern Africa has been shown to have a high rate or oesophageal cancer, with South Africa having one of the highest in the world (Arnold et al., 2015). In South Africa, oesophageal cancer is the 13th and 11th most diagnosed cancer for woman and men respectively, however, is the 5th and 4th most deadly cancer respectively (GLOBOCAN 2020). This is thought to mainly be caused by the high amount of tobacco and alcohol use in the country, as well as poor diet (Alaouna et al., 2019). The two most prevalent types of oesophageal cancer are oesophageal 12 adenocarcinoma and oesophageal squamous carcinoma. Oesophageal adenocarcinoma is commonly found in developed countries where it makes up to 30-50% of the oesophageal cancer diagnoses in these regions (Alaouna et al., 2019). This cancer mainly affects the lower third of the oesophagus. Oesophageal squamous carcinoma is found in the upper oesophagus and is more commonly found in developing countries where it makes up over 95% of all oesophageal cancer diagnoses (Alaouna et al., 2019). The largest risk factors for these cancers are environmental which include consuming alcohol, smoking cigarettes and inhaling or ingesting carcinogenic pollutants (Alaouna et al., 2019; Arnal et al., 2015; Brown et al., 2001). Most of these seem to be due to single nucleotide polymorphisms (SNPs) which cause these detrimental genetic issues (Alaouna et al., 2019). Late-stage oesophageal cancer is also prone to metastasis and treatment can therefore be very difficult if it is not detected at an early stage. Metastasis is the process in which cancer cells migrate and invade distant tissue sites, most commonly through blood vessels or the lymphatic system. The cells can accomplish this by overexpressing certain cell signalling and adhesion proteins, such as cadherins, integrins, CD44 and the 37-kDa/67-kDa laminin receptor precursor/laminin receptor (LRP/LR) (Berno et al., 2005; Geiger & Peeper, 2009). These proteins allow the cells to adhere to different tissues through cell-cell interactions. In the case of LRP/LR the modification of laminin-1 and subsequent proteolytic enzyme activity results in the invasion of new tissue types through the breakdown of the extra cellular matrix (Berno et al., 2005). This marks late-stage cancer (stage 4) and unfortunately, like most cancers, there are no-currently available, dedicated treatment options and therefore conventional treatments must be used. These include chemotherapy, surgery, and radiation therapy. These treatment options, however, come with their own risks and severe side effects. These side effects can include vomiting, diarrhoea, nausea, and immune system suppression. Although chemotherapy disproportionately affects cancer cell mechanisms, such as high proliferation rate and metastasis-enhancing proteins, it’s non-specific nature results in a dose limiting toxicity which limits its overall effectiveness (Malhotra & Perry, 2003). Furthermore, due to its limited oral bioavailability, it is mostly administered intravenously. This is a very non-specific treatment route and can therefore reach and affect more healthy cells. For example, antimetabolites are S-phase specific drugs 13 which interfere in the synthesis of DNA and RNA, whereas DNA intercalating agents such as bleomycin result in DNA strand disruption via oxidative damage. Alkylating agents on the other hand, such as platinum complexes, reduce cell proliferation by creating DNA crosslinks, which prevent the synthesis of RNA and subsequently proteins (Malhotra & Perry, 2003). Chemotherapeutic agents such as these all affect healthy cells and are often used in combination to improve therapy efficacy; however, this can therefore also broadly affect healthy cells in the same way (Corrie, 2007; Lawenda et al., 2008; Malhotra & Perry, 2003). The difficulty in treating cancer, as well as the inadequate current treatment options, makes finding alternative therapies very important, one possible target, due to its interaction with LRP/LR and its involvement in cancer and a variety of age-related disorders could be the enzyme telomerase. 1.2.2. The role of telomeres in age related disorders Cardiovascular Disease (CVD) is a very common age-related disease that resulted in the deaths of approximately 18.6 million in 2019 (Roth et al., 2020). This makes CVD one of the world’s leading causes of death. CVD is a major concern in the developed world as well as the developing world, where health care facilities may not be able to address the issue due in part to the cost involved (Bentzon et al., 2014). CVD refers to a group of progressive diseases that are characterised by high blood pressure, atherosclerosis and myocardial infarctions (Roth et al., 2020). These symptoms are primarily caused by the occlusion (either total or partial) of major and minor blood vessels. This can be brought on by both genetic and lifestyle factors such as suffering from diabetes, obesity as well as eating and exercising habits (Roth et al., 2020). Due to these conditions, the body can experience an increase in ROS, which is one of the principle driving factors of atherosclerosis. Atherosclerosis is the formation of plaques within blood vessels. These plaques can lead to the partial or total blockage of the vessels. Further, the plaques may break apart, creating clots that could migrate to other parts of the body, most notable the brain and heart (Bentzon et al., 2014). These plaques are the result of an increase in ROS which in turn oxidises low density lipoproteins (LDL) causing the formation of oxidised low-density lipoprotein 14 (oxLDL). This causes an increase in the inflammatory process which recruits monocytes (Mallat & Tedgui, 2000). These then differentiate into macrophages which upregulate LOX-1 in an attempt to remove the oxLDL (Li et al., 2004; Pirillo et al., 2013). This leads to the shortening of telomeres and the subsequent activation of apoptosis, which creates foam cells. These foam cells then accumulate and are covered by fibrous tissue from the blood vessel, forming plaques. The plaques subsequently lead to blood vessel occlusion and high blood pressure due to the heat pumping harder to compensate for the blood vessel occlusion (Mallat & Tedgui, 2000). Additionally, it has been shown that telomere shortening leads to the weakening and degradation of vascular beds by causing endothelial dysfunction, leading to increased cellular turnover and vascular wall stress (Serrano & Andrés, 2004). 1.2.3. The role of telomeres in neurodegenerative disorders Telomeres have also recently been implicated in Alzheimer’s disease pathology (Bignoux et al., 2019; Franco et al., 2006; Wang et al., 2015). It has been found that Alzheimer’s disease patients have shorter neuronal telomeres than their health counterparts (Franco et al., 2006). Additionally, further interaction with key Alzheimer’s disease related proteins such as Amyloid- Beta (Aβ)42, shows that telomerase is part of the disease pathology. This is clear, as Aβ42 has been shown to inhibit the functioning of telomerase through the blocking of TERC, the RNA template component (Wang et al., 2015). This would lead to shortening of telomeres and increase tissue deterioration. Furthermore, LRP::FLAG overexpression in HEK-293 and SH-SY5Y cells has also been shown to decrease Aβ shedding and concomitantly intracellular Aβ levels, which may be due to LRP::FLAG promoting TERT expression, as LRP::FLAG overexpression also resulted in an increase in telomerase, which in turn serves a protective role against Alzheimer’s disease pathology by protecting neuronal cells against Aβ induced apoptosis (Bignoux et al., 2019). 15 1.3. Telomerase modulators There are multiple compounds and proteins that are able to modulate telomerase activity, including inhibitors such as metformin and MST-312 and enhancers such as TA-65. These are an interesting target for research, as they may help treat a variety of age-related diseases by either increasing or decreasing telomerase activity. 1.3.1. Metformin inhibits telomerase activity Metformin is an interesting case-study in potential repurposing of existing drugs to combat diseases outside of its original scope. Metformin (1,1-dimethylbiguanide hydrochloride) is an antidiabetic drug for type-2 diabetes sufferers. Although the exact mode of action is not entirely known, the drug is known to activate the AMP-activated protein kinase (AMPK) pathway in conjunction with the protein kinase: liver kinase B1 (LKB1). This leads to an increase in glucose uptake in muscle cells through the phosphorylation of target of rapamycin complex 2 (TORC2) and the subsequent blocking of gluconeogenesis (Vallianou et al., 2013). This helps mitigate insulin resistance in type-2 diabetes sufferers (Zakikhani et al., 2006). Studies have shown that diabetic patients using metformin seem to have lower incidences of hepatocellular, prostate, ovarian, breast, pancreatic and lung cancer (Evans et al., 2005; Vallianou et al., 2013). Additionally, it is known that LKB1 has a tumour suppressor function, as the activation of AMPK by metformin causes a signal cascade, which leads to the suppression of the electron transport chain. Cells therefore compensate by activating p53 in order to arrest cell proliferation to await the restoration of normal glucose levels (Buzzai et al., 2007). This has multiple possible implications, for one it causes p53 negative tumour cells to be unable to resist glucose depravation, severely affecting cell viability (Buzzai et al., 2007) and therefore for p53 positive tumour cells, it may aid in the activation of the p53 DNA damage pathway. This could indicate 16 that the drug may be able to decrease cell proliferation and therefore potentially reduce the risk of cancer. Indeed, studies have shown that individuals using metformin displayed reduced cancer risk (Evans et al., 2005; Tseng, 2019; Vallianou et al., 2013). Additionally, it has been shown that cell proliferation in oesophageal squamous carcinoma cell lines is significantly reduced in the presence of metformin (Damelin et al., 2014). Furthermore, it was demonstrated that if mice were treated with metformin in conjunction with conventional chemotherapy, they experienced longer remission times compared to the untreated mice (Hirsch et al., 2009). Metformin treatment also resulted in a decreased recurrence for gastric cancer patients after surgery (Lee et al., 2015). Previous research has shown that metformin not only seems to have an anti-proliferative effect on cancer cells, but it seems to affect telomere dynamics, including telomerase activity, which is involved in cancer progression (Bernert et al., (unpublished)). Moreover, hTERT mRNA levels have been shown to be reduced by metformin in endometrial cancer, however, the mechanism is not yet understood (Hanna et al., 2012). Our previous research showed a significant reduction in telomerase activity after metformin treatment in an oesophageal cancer cell line expressing wild type p53 (Bernert et al., (unpublished) supplementary Figure 7.1). Telomerase activity was assessed using the TRAPeze RT telomerase activity assay (as described by Bignoux et al., 2023 and in section 3.1.8.1). One possible explanation put forward by Kanaya et al. is that p53 represses hTERT expression by acting as a transcription factor and binding the SP1 binding sites found in the hTERT promoter. This was shown by overexpressing p53 in SiHa (cervical carcinoma) cells via adenoviral infection, resulting in a decrease in hTERT expression and a subsequent decrease in telomerase activity (Kanaya et al., 2000). Since the telomerase component, hTERT, is known to be transported to the mitochondria via its mitochondrial leader sequence, and that metformin acts within the mitochondria (Buzzai et al., 2007), there may be a direct interaction between the drug, hTERT and p53 which affects cancer progression. 17 1.3.1. The MST-312 telomerase inhibitor Epigallocatechin gallate (EGCG), a tea derived catechin/antioxidant, was found to inhibit telomerase directly (Seimiya et al., 2002). However, the fact that this compound is a natural product and is therefore difficult to synthesise and produce in large quantities, lead to the creation of simpler synthetic derivatives including MST-312 (([N,N’-bis(2,3-dihydroxy-benzoyl)- 1,2-phenylenediamine]) (Seimiya et al., 2002). MST-312 was especially effective at inhibiting telomerase activity, with a determined IC50 (50% inhibitory concentration) of only 0.67 µM and an effective dose of 1-2 µM, however, the mechanism by which it accomplishes this is so far unknown (Seimiya et al., 2002). MST-312 proved to be easily synthesised in large quantities and may be an interesting candidate as a novel chemotherapeutic agent. 1.3.2. The TA-65 telomerase enhancer TA-65 is a compound isolated from the root of Astragalus membranaceus, a plant used in traditional Chinese medicine (de Jesus et al., 2011; Harley et al., 2011). This compound has been studied in humans as a dietary supplement and has been found to significantly decrease the percentage of cells with short telomeres (<4 kbp) without negative side effects (Harley et al., 2011). This was confirmed in mice, where significant telomere lengthening was seen without a concomitant incidence of cancer (de Jesus et al., 2011), as well as in cell culture, where TA-65 increased telomerase activity in foetal fibroblasts and neonatal keratinocytes two- to threefold at a 30 nM concentration (Harley et al., 2011). Telomerase activity plays an integral part in various diseases, which make telomerase modulators an interesting prospect for the treatment of these diseases in conjunction with traditional 18 treatment options. One such telomerase modulator, which has been shown to enhance telomeres activity is LRP/LR. 1.4. 37 kDa laminin receptor precursor/67 kDa high affinity laminin receptor 1.4.1. Structure and function The 37 kDa Laminin Receptor Precursor/67 kDa High Affinity Laminin Receptor (LRP/LR) has recently been shown to influence telomerase activity (Naidoo et al., 2015; Otgaar et al., 2017). LRP/LR is a highly conserved non-integrin transmembrane receptor, which is able to bind a variety of molecules such as prion proteins, elastin and laminin (Gauczynski et al., 2001, 2006; Mercurio, 1995) and is implicated in a variety of disorders (Figure 1.7). Although it performs its major functions on the cell surface, such as cell migration and invasion of foreign tissues, LRP/LR has been found in the cytosolic domain as well as the perinuclear compartment, suggesting that it may play a role in many additional processes, such as cell proliferation and growth (Gauczynski et al., 2001; Naidoo et al., 2015). Similar to TERT, LRP/LR has been shown to influence cell survivability and proliferation, particularly in cancer cells. This can be seen by the fact that LRP/LR is not only involved in tissue differentiation, but in adhesion and invasion of cancer cells through the binding of laminin-1 (Chetty et al., 2014; Jovanovic et al., 2015; Mercurio, 1995). Since LRP/LR is so widespread within the cell, some additional functions of the receptor are nuclear structure maintenance as well as translational functions (Jovanovic et al., 2015). 19 Figure 1.7: Schematic representation of the LRP/LR transmembrane receptor, binding various biomolecules The function of LRP/LR as a as well as common binding targets are depicted with the N-terminus of the receptor found in the cytosol. This is then connected to the short transmembrane domain. The C-terminus is shown outside of the cell in the extra cellular matrix, where molecules bind to the active sites of the receptor to elicit various cellular responses, including IgG antibody binding at the 272-280 binding site. LRP/LR has been shown to bind primarily laminin-1 and heparin sulphate proteoglycans (HSPs) in the 205- 229 binding site Laminin-1, HSPs and cellular prion proteins (PrPc) in the 161-180 binding site. (Jovanovic et al., 2015). 1.4.2. LRP/LR and telomerase This proximity to the nucleus puts LRP/LR in very similar locations to TERT and these proteins may therefore have an interaction. This is indeed the case, as it has been shown that LRP/LR 20 increases telomerase activity to promote cell viability and prevent cellular senescence as shown by the reduction of gamma H2AX (which marks sites of DNA damage and DNA breaks) and the senescence biomarker β-galactosidase (Bodnar et al., 1998; Otgaar et al., 2017). Further evidence shows that hTERT protein levels, telomerase activity and subsequently telomere length were increased after the overexpression of LRP::FLAG within both the MRC5 and HEK-293 cell lines. This may suggest that LRP/LR or LRP::FLAG aids in the formation of the active telomerase enzyme from its major hTERT and hTERC components. This is evidenced by their co-localisation as well as telomerase activity increasing with increased LRP levels (Bignoux et al., 2019; Bodnar et al., 1998; Cuttler et al., 2020; Otgaar et al., 2017). This interaction has potential applications for the treatment of various diseases including cancer and neurodegenerative diseases. 1.4.3. The role of LRP/LR in cancer Due to the highly conserved nature of LRP/LR across a multitude of species, it has become a common component in disease pathogenesis. Most notably for cancer and neurodegenerative diseases. As previously mentioned, LRP/LR is involved in cell proliferation, survivability, and migration. This has led LRP/LR to be overexpressed in tumourigenic cells, in order to overcome their replication limit and become metastatic (Chetty et al., 2014; Jovanovic et al., 2015). In addition to this, the receptor has been found to be involved in the promotion of angiogenesis and causes a reduction in apoptosis (Chetty et al., 2013, 2014, 2015). Angiogenesis relies heavily on the prior degradation of the basement membrane before the restructuring of the endothelial cells into vascular structures. It is theorised that LRP/LR helps facilitate this process through its laminin-1 interactions, by activating metalloproteases such as collagenase (Chetty et al., 2013). Additionally, LRP/LR is thought to bind chromatin and the nuclear envelope to one another during interphase of the cell cycle. This is thought to result in increased chromosomal stability and cell viability, leading to a bypassing of apoptosis (Chetty et al., 2015). Consequently, it was also shown that when LRP/LR was blocked using an anti-LRP/LR specific antibody (IgG1-iS18) or downregulated by the LRP-targeting siRNA, that adhesion, invasion, angiogenesis, and cell 21 viability were decreased in cancerous cells (Chetty et al., 2013, 2015; Jovanovic et al., 2015; Naidoo et al., 2015). The newly discovered link between LRP/LR and telomerase also supports this trend, as it was shown that by overexpressing LRP::FLAG, hTERT levels increased and telomerase activity increased, which could lead to cell immortalisation and increase cell viability (Bignoux et al., 2019; Naidoo et al., 2015; Otgaar et al., 2017). These interesting interactions with LRP/LR could lead to the development of novel anti-cancer treatments in the future through the downregulation or blocking of the receptor protein. 1.4.4. The role of LRP/LR in neurodegenerative disease Similarly, LRP/LR has been shown to play a significant role in neurodegenerative diseases such as Alzheimer’s disease and prion disorders. In prion diseases, LRP/LR has been shown to be a receptor for the scrapie prion protein (PrPSc), which facilitates the internalisation of the prion, leading to aggregation and an eventual toxic build-up resulting in cell death termed spongiform encephalitis (Gauczynski et al., 2006; Leucht et al., 2003). In Alzheimer’s disease, LRP/LR has been shown to promote Aβ shedding and internalisation. This can lead to aggregation and cytotoxicity within neuronal cells, driving the formation of amyloid plaques (da Costa Dias et al., 2013, 2014; Jovanovic et al., 2015). Similarly, to cancer, the blockage or downregulation of LRP/LR by IgG1- iS18 and LRP-targeting siRNA, respectively, has been shown to improve the disease state via reducing cytotoxicity, by decreasing Aβ aggregation as well as improving cell viability (da Costa Dias et al., 2013, 2014; Jovanovic et al., 2015; Leucht et al., 2003). Additionally, LRP::FLAG has been shown to have a positive effect on the Alzheimer's disease state. After the transfection and subsequent overexpression of LRP::FLAG in HEK-293 and SH-SY5Y cells, LRP::FLAG was shown to directly interact with the tau protein (Cuttler et al., 2020). This overexpression further showed a decrease in PrPc levels as well as phosphorylated tau. Briefly, the tau protein is associated with and helps stabilise the assembled microtubules. In Alzheimer’s disease, however, the tau protein is hyperphosphorylated and dissociates from the microtubules. These loose tau proteins then associate with one another and eventually form neurofibrillary 22 tangles that lead to cell death (Haass & Mandelkow, 2010). Furthermore, LRP::FLAG overexpression in HEK-293 and SH-SY5Y cells has also been shown to decrease Aβ shedding and concomitantly intracellular Aβ levels (Bignoux et al., 2019), similar to what was observed after downregulating LRP/LR. This may be due to LRP::FLAG promoting TERT which in turn serves a protective role against Alzheimer’s disease pathology (Bignoux et al., 2019). This further shows that anti-LRP/LR drugs could be very useful in combating neurodegenerative diseases, however, transfection is not a viable treatment option, as it might cause off-target effects, and so a different approach is needed. Using the LRP protein alongside a non-toxic drug delivery system would mitigate these issues. 1.5. Applications of nanoparticles in drug delivery and research Polymer and metallic nanoparticles have become an interesting prospect for both the delivery of therapeutic agents, due to their low toxicity and biocompatibility, as well as for the use as a research tool, due to their interesting optical properties. 1.5.1. PLGA nano-capsules as drug delivery agents Nano-capsules, such as those synthesised using Poly(lactic-co-glycolic acid) (PLGA), are able to contain the active compound of a drug. They can contain anything from small chemical molecules, nucleic acids and plasmids to proteins (Danhier et al., 2012; Gholizadeh et al., 2022). The molecules contained within the nanoparticles also gain the benefit of having increased stability compared to those that are not protected, allowing them to have longer active periods compared to when unprotected (Sharma et al., 2016). PLGA nanoparticles are also biodegradable, as natural processes are able to hydrolyse the polymer into its constituent monomers: lactic acid and glycolic acid, which are easily metabolised (Danhier et al., 2012; 23 Sharma et al., 2016). This makes it more useful than functionalised metallic nanoparticles, as these could build up within a system to toxic levels due to them not being able to be metabolised. In the case of PLGA, this polymer has previously been used in medical procedures as biodegradable (dissolvable) sutures (Mccall & Sirianni, 2013), making PLGA a safe and tested compound in medical applications. PLGA nanoparticles enter the cell by endocytosis, after which they leave the endo-lysosome and are released into the cell (Vasir & Labhasetwar, 2007). PLGA nano-capsules can also be further modified to target specific regions of the body or even specific cell types. This can be done through PEGylation, where polyethylene glycol (PEG) is added to the surface of the nano-capsule (Danhier et al., 2012). PEG is a widely used polymer which can bind a variety of molecules such as proteins and antibodies. Specific cell surface receptors could be targeted via surface antibody functionalisation, increasing the uptake of the therapeutic in desired tissues. This is very beneficial to the treatment of specific disorders, as there would be an increase in drug availability for these tissue sites and at the same time, there would be less off-target effects due to less uptake of the therapeutic in non-targeted tissues. This has potential applications in many different diseases, for example, by bypassing obstacles such as the blood brain barrier they can be used to treat neurodegenerative diseases (Hoyos-Ceballos et al., 2020). In cancer treatment on the other hand, it would be beneficial if the drug specifically affected only the cancer cells, unlike chemotherapy, which affects healthy cells as well (Lawenda et al., 2008). This would improve the quality of life for patients suffering from severe chemotherapy-related side-effects. 1.5.2. Nanoparticles in telomerase activity assays Telomerase is an important target for cancer research, and therefore many different telomerase activity assays have been developed. These are often based on the telomeric repeat amplification protocol (TRAP). A TRAP protocol consists of a DNA telomerase substrate which is amplified by the telomerase enzyme in the presence of dNTPs (reviewed in Zhou & Xing, 2012). After a whole protein extraction process, the active telomerase enzyme extends the telomeric repeats on the 24 DNA substrate, which can be quantified by a quantitative polymerase chain reaction (qPCR) (as described in section 3.1.8.2) or conventional PCR. The number of repeats added is directly proportional to the signal obtained, which is then an accurate, relative measurement of telomerase activity. TRAP based telomerase activity assays are usually reliable, however, they can present many different problems. These protocols require whole protein extract from tissues or cells, which can leave impurities behind. Due to the sensitivity of the DNA binding dyes and fluorescent probes utilised by these protocols, any impurities, including cell debris, genetic material and trace RNases can adversely affect the experiment. This could cause interference with the RNA component of telomerase, or DNA contamination, non-specific binding and the formation of primer dimers could lead to false positives. This particularly affects telomerase activity assays, as they mainly quantify the extension of DNA (reviewed in Zhou & Xing, 2012). qPCR based techniques are also often expensive and time consuming. Therefore, creating a need for alternative, more cost-effective assays with fewer obstacles. Metallic nanoparticles, for example gold nanoparticles (AuNPs) are another type of nanoparticle that have very interesting optical properties. AuNPs change the colour of the nanoparticle colloid solution based on their size and proximity to one another (Yarbakht & Nikkhah, 2016). The larger the AuNPs are in solution (20-90 nm in diameter), the greater the wavelength of the peak absorbance of the nanoparticle solution (>600 nm absorbance, more blue). On the other hand, the smaller the AuNPs in the solution (10-20 nm in diameter), the lower the wavelength of the peak absorbance of the nanoparticle solution (500-600 nm absorbance, more red). If AuNPs become more aggregated, the colour of the solution also shifts towards blue as they are mimicking larger nanoparticle sizes. This colour shift will continue until the gold aggregates become macro in scale. This aggregation can be achieved by adding salt to the AuNP solution, which causes the AuNPs to stop repelling each other and begin to interact with one another. This occurs due to a change in surface charge, which can be exploited to surface modify the nanoparticles. Due to these properties, one could very easily detect changes to the surface of the nanoparticle, as even a small change could result in an observable and therefore easily spectrophotometrically measurable, colour change (Yarbakht & Nikkhah, 2016). This sensitivity as well as their ability to be easily functionalised to many different molecules, including the 25 covalent bonding of thiolated DNA to the surface of the AuNP, could make AuNPs a very useful biosensor in detecting telomerase activity. If AuNPs could be coupled to a telomerase substrate (thiolated DNA), and salt is added to the solution, the colour of the solution would shift towards blue due to the close association of the particles. However, once telomerase is allowed to elongate the substrate, the steric hindrance caused by the elongated DNA, and possible secondary structure formation, will prevent the nanoparticle from closely associating. These secondary structures are caused by the extended telomerase substrate folding in on itself and subsequently preventing the close association of the AuNPs. Since the modified AuNPs now resist aggregation, this would cause the colour of the solution to shift from a blue to a more red colour (Wang et al., 2012) (Figure 1.8). This colour change could then be measured using UV-visual spectrophotometry (Wang et al., 2012). Detecting telomerase activity using nanoparticles could prove very beneficial, as many steps found in conventional qPCR-based methods could be eliminated. This includes the use of fluorescent probes and dyes, which can easily degrade, be quenched, auto-fluorescence and show false positives due to primer dimers, resulting in differing results from one experiment to another. This would also mitigate signal bleed through in 96-well plates, which can result in false positive results. Nanoparticles can reduce the time and cost involved in performing telomerase activity assays, due to the reduced need for specialised equipment. In addition, the simplicity of the reaction lends itself to shorter preparation times, while still maintaining the sensitivity of the assay. Indeed, telomere extension has been detected in the protein extract of only 10 HeLa cells using AuNPs (Wang et al., 2012). Here, Wang et. al successfully confirmed a reduction in telomerase activity, using the TMPyP4 telomerase inhibitor, with their gold nanoparticle assay (Wang et al., 2012). If this concept could be adapted for rapid telomerase activity analysis, it would become a valuable tool for the research of age-related diseases which heavily involve telomerase. 26 Figure 1.8: Principles of a AuNP-based telomerase activity assay Thiolated DNA (telomerase substrate) is functionalised to AuNPs. After functional telomerase is introduced to the nanoparticle solution, the substrate is elongated in a PCR-like reaction. This causes steric hindrance between the particles, causing them to disperse and causes a red shift in colour. The nanoparticle solution with the non-elongated substrate turns a blue colour due to the close association of the nanoparticles (Wang et al., 2012). 1.6. Research rationale Telomere dynamics, specifically telomerase activity, have become a vitally important factor when studying a variety of age-related diseases, such as CVD and cancer. This makes the accurate detection of telomerase activity within cell cultures and tissue samples a necessity. Conventional techniques have many drawbacks, including their very high cost. This project, therefore, aimed to develop a AuNP based assay to determine telomerase activity. This would fulfil the need for an accurate, cost-effective tool to determining telomerase activity in a research capacity. This assay could be used for the research of a variety of diseases, such as cancer. It is important to 27 note, however, that this assay would not be limited to cancer but can easily be used in any age- related disease field or as a general telomerase activity assay. As stated above, previous research has shown that telomerase activity is directly affected by the LRP protein, which has been shown to have therapeutic effects in Alzheimer’s disease models. The AuNP-based telomerase activity assay could be used to assess this effect on telomerase activity by an LRP-based drug while telomerase inhibitors, such as MST-312, can be used to test its sensitivity. The development of an LRP-based drug could therefore be very beneficial, since the nano-capsule would protect the therapeutic agent from degradation and can facilitate the safe uptake into cells without being degraded via natural immune responses. Additionally, as LRP is a natural protein found in the body and that no additional non-degradable or cytotoxic substances are taken in by the cells, the elevation of LRP levels in this manner should not adversely affect cell viability. Due to the wide range of applications elevating LRP levels has in the treatment of different disorders, this could represent a safer alternative to plasmid transfection treatment and could potentially be used for the treatment of age-related diseases, including Alzheimer’s disease, through its ability to increase telomerase activity. 28 2. Aims and Objectives 2.1. Aim 1: Optimise the gold nanoparticle telomerase activity assay and improve the stability of the functionalised AuNPs by redesigning separate linker and extension strands. 1. Synthesise AuNPs using the chemical hydrothermal method. 2. Improve DNA functionalisation by employing pH-dependent binding methods. 3. Improve signal acquisition by optimising sample volume and utilising spectrum analysis to determine the optimal acquisition wavelengths. 4. Treat oesophageal cancer cells with metformin and the MST-312 telomerase inhibitor to detect changes in telomerase activity. 5. Compare conventional telomerase activity assays to the AuNP-based telomerase activity assay. 2.2. Aim 2: Create an LRP based nanoparticle drug. 1. Optimise PLGA nano-capsule synthesis by the emulsion solvent evaporation technique. a. Test various synthesis protocols, solvents, and emulsifiers. b. Optimise purification of the nano-capsules while retaining high yields. 2. Characterize PLGA nano-capsules using UV-visual spectrophotometry, dynamic light scattering and scanning electron microscopy. 3. Determine cytotoxicity profile of empty PLGA nano-capsules on HEK-293 cells. 4. Synthesise and purify the therapeutic protein LRP. 5. Encapsulate the LRP protein using PLGA. 6. Confirm encapsulation and cytotoxicity profile of the LRP-encapsulated PLGA nano- capsules on HEK-293 cells. 7. Assess the effect on telomerase activity of both empty and LRP-encapsulated PLGA nano- capsules on HEK-293 cells. 29 3. Materials and Methods 3.1. Cell culture 3.1.1. Cell culture protocol Oesophageal carcinoma cell line; WHCO-5 (Veale & Thornley, 1989), and human embryonic kidney cells; HEK-293 (ATCC, USA) (Graham et al., 1977), were cultured for this project. The HEK- 293 cells have previously been used to optimise procedures involving telomerase activity and TERT expression, as these cells are known to display relatively high levels of TERT and telomerase activity (Letsolo et al., 2010). Ethics approval for these cell lines was obtained from the Human Research Ethics Committee (Medical): reference number W-CJ-140804-1. Each of the cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Hyclone, GE Life Sciences, Massachusetts, USA) containing 10% Foetal Bovine Serum (FBS) (Biowest, Nuaillé, France) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific, Massachusetts, USA). The cells were kept in a 5% CO2, 37 °C humidified atmosphere to ensure that the pH of the system remained constant through the CO2/HCO3- buffering system and that the cells experienced maximum cell growth by mimicking in vivo like conditions. 3.1.2. Passaging of cells Each cell culture flask was viewed under an inverted light microscope (Zeiss Primovert) to monitor cell growth, cell detachment and to determine the level of confluency. After the cells reached approximately 80% confluency, they were harvested and passaged to prevent contact inhibition and therefore allow the cells to continue growing. To passage the cells, the cells were first detached from the flask using trypsin-EDTA treatment. The trypsin-EDTA (Biowest, Nuaillé, France) was then inactivated by adding an equal volume of supplemented cell culture medium. 30 The cells were evenly passaged into multiple T25 culture flasks (NeST Technology, Virginia, USA) at a density of 0.7x106 cells/ml. 3.1.3. Cryo-preservation of cells After harvesting, cell samples were cryopreserved to be re-cultured at a later stage. After trypsinisation the cells were harvested and centrifuged at low speed (±200-400 g) for 10 minutes. The excess media was then removed, and the pellet resuspended in a cryopreserving solution (15% Glycerol (Associate chemical enterprise, Southdale, South Africa), 20% FBS, 65% DMEM). The vials were then kept at -20 ˚C overnight and transferred to -80 ˚C or liquid nitrogen storage. When thawing the frozen cells, an initial high FBS concentration of 20% was used to provide extra nutrients for the cells. This was then stepped down to 10% once the cells stabilised. 3.1.4. Cell quantification To accurately quantify the number of cells in each flask once they reached confluency, a Neubauer haemocytometer or the TC 20™ automated cell counter was used. The cells needed to be quantified to ensure subsequent experiments and subcultures utilise consistent numbers of cells for reliable results. To distinguish between dead and live cells, the trypan blue stain was used. This stain is only taken up by dead cells due to their porous/permeable cell membrane and is excluded by live cells due to their intact membrane. This leads to dead cells being stained blue under the microscope (Tran et al., 2011). This allows for easy quantification of unstained, live cells. The cell suspension (20 µl) was mixed with 20 µl trypan blue for a 2x dilution and a small sample added to the Neubauer haemocytometer. Live cells were then quantified, using the 16- square region of the haemocytometer, while utilising a light microscope (Zeiss Primovert). Thereafter, cell concentration, total number of cells as well as cell viability were calculated using the following formulas: 31 𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 = 𝐶𝑒𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ( 𝑐𝑒𝑙𝑙𝑠 𝑚𝑙 ) 𝑥 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛 𝐶𝑒𝑙𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 ( 𝑐𝑒𝑙𝑙𝑠 𝑚𝑙 ) = 𝑐𝑒𝑙𝑙𝑠 𝑝𝑒𝑟 16 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑥 104 x dilution factor 𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = ( 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠 𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 ) 𝑥 100 3.1.5. Metformin and MST-312 Treatments Metformin treatment: A 250mM filter sterilised metformin stock solution was created using the above-mentioned culture medium. Thereafter flasks were seeded with newly passaged cells. The cells were allowed to attach for 24 hours before they were treated with metformin (Merck, Darmstadt, Germany). Each flask was then treated with 10 mM metformin and incubated for 48 hours alongside the untreated controls. The cells were then harvested as mentioned above and utilised in subsequent downstream procedures. MST-312 Telomerase inhibitor treatment: A 10 mM filter sterilised MST-312 (Thermo Fisher Scientific, Massachusetts, USA) stock solution was created using the above-mentioned culture medium. Thereafter flasks were seeded with newly passaged cells. The cells were allowed to attach for 24 hours before they were treated with the inhibitor. Each flask was then treated with a final concentration of 4 µM MST-312 and incubated for 48 hours alongside the untreated controls. The cells were then harvested as mentioned above and utilised in subsequent downstream procedures. 32 3.1.6. MTT cell viability assay The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Thermo Fisher Scientific, Massachusetts, USA) assay was used to determine cell viability of the treated cells. A 5 mg/ml MTT stock solution was created using serum free cell culture media. The MTT stock solution was then filter-sterilised and added to the cells (in serum free culture media) to a final concentration of 0.5 mg/ml. Only viable cells reduce the reagent through cellular NAD(P)H- dependent oxido-reductases, as these enzymes are involved in metabolic activity. This reaction is carried out for 4 hours as the viable cells reduce the yellow MTT reagent into an insoluble formazan product, which is purple in colour. This is a reflection of cell viability. The precipitate is then treated with a detergent such as DMSO (Associate chemical enterprise, Southdale, South Africa) and mixed until fully dissolved. After the formazan crystals are dissolved, the absorbance is measured at 570 nm, using a spectrophotometer (VICTOR® Nivo™ Multimode Microplate Reader - PerkinElmer). The more viable cells that are present (the more active enzyme), the higher the amount of dissolved product and therefore the higher the absorbance reading. This assay was performed in 96-well plates (NeST Technology, Virginia, USA) to a final volume of 200 µl, where each well was seeded with 5000 cells. The cells were then allowed to attach overnight, and treatment commenced the following day. Each treatment as well as each control was performed in biological and technical triplicates. The controls included the no-cell control, no-MTT control, 100% dead cells (treated with 1% triton x-100 - Biorad Hercules, CA, USA) as well as the untreated cell control. The absorbance readings of the treated samples were then compared to those of the controls which were set to 100% cell viability to more easily compare. 3.1.7. DNA quantification - agarose gel electrophoresis DNA was resolved on 1.5% TBE agarose gels (Thermo Fisher Scientific, Massachusetts, USA) at 90V for 45-60 minutes for the DNA to adequately separate. This method utilises the negative 33 charge of the DNA as well as the pore size of the agarose gel to separate DNA fragments according to their size. Each sample was resolved alongside a 1 kbp DNA ladder (New England Biolabs, Ipswich, MA, USA) containing known fragment lengths. Through the application of an external electric field, the negatively charged DNA is repelled by the negatively charged electrode and is forced through the pores of the gel. The shorter the fragment, the more quickly it travels, leading to a separation of size. After the addition of GR-Green (Inqaba Biotechnical Industries, South Africa), this separation can be seen under UV light as bands in the gel. The integrity of the DNA was determined by observing the streaking patterns on the gel. The less streaking, the more intact the DNA. 3.1.8. Telomerase activity 3.1.8.1. TRAPeze Assay The TRAPeze® RT Telomerase Detection Kit (Merck, Darmstadt, Germany) was used to determine telomerase activity for all cell lines. This assay represents a common, accurate telomerase activity assay which was used to compare to the novel AuNP-based telomerase activity assay. qPCR amplifies a target DNA strand like conventional PCR but is also able to simultaneously quantify the synthesis of the DNA strand. With the TRAPeze kit, this was achieved using a sequence specific primer/DNA probe containing a fluorescent reporter as well as a quencher molecule. These reporter molecules fluoresce; however, due to the proximity of the quencher molecule, the fluorescent signal is suppressed. After the telomerase extraction from mammalian cells, the enzyme elongates the telomerase substrate, and the probe causes a complimentary strand to be synthesised by hot start taq polymerase. The incorporation of the probe causes the distance between the quencher and the fluorescent reporter molecule to increase, creating a fluorescent signal. Therefore, the fluorescent signal is proportional to the amount of added telomeric repeats. Protein, including telomerase, was extracted from cell pellets using 3-[(3- Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (Merck, Darmstadt, Germany) lysis buffer as it is non-denaturing and therefore capable of extracting active enzymes. 34 Cells are first harvested via trypsinisation, washed in PBS and the resulting cell pellets are each lysed in 200 µl CHAPS lysis buffer for 30 minutes on ice. The cell lysate is then centrifuged at 12 000 xg for 20 minutes at 4 °C. The protein-containing supernatant was harvested, and the amount of protein being used was standardised to 0.5 mg/ml (final concentration of 50 ng/µl). The HEK- 293 cells were used as a positive control for telomerase activity as they display elevate telomerase activity (Letsolo et al., 2010). Additionally, heat treated HEK-293 cells were used as negative telomerase controls, as the heat treatment inactivated the enzyme. CHAPS and no- template-controls were also prepared to account for possible buffer effects. The following thermal cycle was used on the Roche LightCycler LC480 (Roche, Basel, Switzerland): Pre- incubation consisted of one cycle of 37 ˚C for 30 minutes and 95 ˚C for 2 minutes. Amplification consisted of 45 cycles of 95 ˚C for 30 seconds, 59 ˚C for 1 minute and 45 ˚C for 10 seconds (where a single acquisition, fluorescent data acquisition, was performed). The readings were compared to TSR8 standard curve (20-0.2 amoles/ µl) and analysed using Agilent Aria Software (v1.5) and expression fold change calculated using the ∆∆Ct method: ∆ 𝐶𝑡 = 𝐶𝑡 (𝑔𝑒𝑛𝑒 𝑜𝑓 𝑖𝑛𝑡𝑒𝑟𝑒𝑠𝑡) − 𝐶𝑡 (ℎ𝑜𝑢𝑠𝑒𝑘𝑒𝑒𝑝𝑖𝑛𝑔 𝑔𝑒𝑛𝑒) ∆∆𝐶𝑡 = ∆𝐶𝑡 (𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒) − ∆𝐶𝑡 (𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑠𝑎𝑚𝑝𝑙𝑒) 𝐸𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛 𝑓𝑜𝑙𝑑 𝑐ℎ𝑎𝑛𝑔𝑒 = 2(−∆∆𝐶𝑡) 3.1.8.2. Modified TRAP assay In addition to the TRAPeze assay, a modified TRAP assay was used to determine telomerase activity. Protein extraction and sample preparation was done as previously described in the above TRAPeze assay section 3.1.8.1. Samples were mixed with a TRAP reaction buffer, consisting of ultra-pure BSA (which acts as a PCR enhancer) (Merck, Darmstadt, Germany), EGTA (Merck, Darmstadt, Germany) (which inhibits RNAses that could interfere with TERC), Luna® Universal qPCR Mastermix (New England Biolabs, Massachusetts, USA), RNAse free water, ACX primer (Inqaba Biotechnical Industries, South Africa) (an anchor that prevents primer dimer formation and therefore self-amplification) and the TS primer (Inqaba Biotechnical Industries, South Africa) 35 (which acts as a telomerase substrate) (Table 3.1). The assay was performed with the following thermal cycle: Pre-incubation consisted of one cycle of 37 ˚C for 1h and 95 ˚C for 2 minutes. Amplification consisted of 45 cycles of 95 ˚C for 30 seconds, 59 ˚C for 1 minute and 45 ˚C for 10 seconds (where a single acquisition, fluorescent data acquisition, was performed). The readings were compared to TSR8 standard curve (20-0.2 amoles/ µl) and analysed using Agilent Aria Software (v1.5) on the Roche LightCycler LC480 (Roche, Basel, Switzerland) as stated above. Table 3.1: Telomerase activity primers Name Primers (5'-3') Annealing Temp (°C) Reference TS Forward AATCCGTCGAGCAGAGTT 58 Kim et al., 1997 ACX Reverse GCGCGGCTTACCCTTACCCTTACCCTA 3.2. Section 1: Gold nanoparticle telomerase activity assay Telomere dynamics, specifically telomerase activity, have become a vitally important factor in a variety of age-related diseases, such as CVD, Alzheimer’s disease, and cancer. Thus, this technology presents a potential research kit utilizing gold nanoparticles to determine the activity of the telomerase enzyme. Through the addition of telomeric repeats and therefore the elongation of the synthetic telomeres attached to the AuNPs, the extracted telomerase leads to a colour change in the gold nanoparticle solution. This colour change is detectable using spectrophotometric readings and represents telomerase activity. This would be useful as an alternative to expensive existing telomerase activity kits for the use in cancer, ageing and age- related disease research. 36 3.2.1. AuNP synthesis A concentrated 3:1 mixture of HCl:HNO3 (aqua regia) (HCL and HNO3 acquired from Associate chemical enterprise, Southdale, South Africa) was used to treat glassware. This prevents nanoparticles from attaching themselves to the sides of the glassware, which prevents unwanted nucleation points that would otherwise res