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Molecular Studies of an Alternative Lengthening of Telomeres (ALT) Mechanism

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Molecular Studies of an Alternative Lengthening of Telomeres (ALT) Mechanism Molecular Studies of an Alternative Lengthening of Telomeres (ALT) Mechanism by Kilian Thomas Perrem A thesis submitted to the University of Sydney in fulfilment of the requirements for the degree of Doctor of Philosophy The Children’s Medical Research Institute Faculty of Medicine University of Sydney March, 2001 Statement of Originality The contents of this thesis have not been presented for the award of a degree at this or any other university. The data are the original work of the author except where indicated. Kilian Perrem ii Acknowledgments This thesis would not have been possible without the scientific, technical and moral support of a large number of people both inside and outside the lab. First and foremost I must thank Roger Reddel for accepting me into his lab and giving me the opportunity to complete a PhD under his expert and diligent, but always good humoured supervision. Thanks also goes to Professor Peter Rowe, Director of the Children's Medical Research Institute, for supporting my PhD candidature and my research and also for allowing me to travel to conferences, both in Australia and overseas, as part of my career development. I must also mention my former mentor, Antony Braithwaite, who employed me as a technician some years ago but always treated me as a colleague. Thank you Antony for starting me on this path. My colleagues in the Cancer Research Group both past and present have been invaluable as fellow scientists and friends in helping me through the highs and lows of my scientific endeavours. I must specifically thank Axel Neumann for all his help and patience in undertaking many cytogenetic analyses which are presented here and for his imaging work. All of the wonderful and numerous FISH data in this thesis are due also to Axel’s expertise. Clare Fasching was equally as generous with her time and experience in chromosomal matters for which I am extremely grateful. I am particularly thankful to Clare for her chromosome transfer expertise which generated the hybrid clones with neo tagged telomeres in Chapter 4 and also for producing Figures 1-1 and 7-3 and for chromosome painting expertise which generated Figure 7-1. Thanks goes also to Tom Yeager for APB immunostaining analysis and for generating the images used in Figure 4-10. Lorel Colgin was always happy to share her wonderful TRAP and RT-PCR assay abilities with me and analyse samples which are shown in Figures 4-7, 5-3 and 6-2. In addition I am grateful to Lorel for her generosity in letting me use her cell lines and clones at will to back up my own work and for contributing some of her own data to this thesis, specifically Figures 5-1 and 5- 2. Thanks goes also to Axel, Lorel and Clare and also Lily Huschtscha and Christian Toouli for proofreading and comments on different sections of this thesis. Part of my work was done in collaboration with Professor Rob Newbold and Dr. Andrew Cuthbert of Brunel University, UK. I thank them and members of their research team, Dr. Deborah Trott and Alison Marriott, for providing their chromosome transfer expertise which enabled us to do some exciting work in our search for ALT repressors which is the subject of Chapter 7. Hopefully this aspect of the work and their collaboration will continue in the future. iii A special thanks to Lindy Hodgkin for all of her help over the years with computer related disasters and for maintaining the reference database with such vigilance. All of the support staff at the CMRI are also to be thanked for providing a working environment for successful research to take place. I am no doubt spoiled forever by the facilities at the CMRI! Particular thanks goes to Christine Smyth for undertaking the flow cytometry analysis shown in Figure 3-2 and help with that data. Finally I must thank my wife Catherine for all of her love, support and encouragement over the past four years. Quite simply, there are numerous things including this thesis which would never have been made possible without her. iv Publications arising from this thesis Perrem K., Bryan TM., Englezou A., Hackl T., and Reddel RR. Repression of an Alternative Mechanism for Lengthening of Telomeres in somatic cell hybrids. Oncogene (1999) 18, 3383- 3390. Perrem K. and Reddel R.R.. Telomeres and Cell Division Potential. Progress in Molecular and Subcellular Biology (1999) Vol 24, 173-184. Springer-Verlag Berlin Heidelberg. Reddel, R.R., Bryan, T.M., Colgin, L.M., Perrem, K.T., and Yeager, T.R. Alternative lengthening of telomeres in human cells. Radiation Res (2001) 155(1), 194-200. Perrem K., Colgin L.M,, Neumann A.A,, Yeager T. R., and Reddel R.R. Expression of telomerase in ALT cells lengthens the shortest telomeres but does not repress ALT. Mol. Cell. Biol. Submitted. v Abstracts Perrem, K., Englezou, A. and Reddel, R.R. A study of an alternative mechanism for lengthening of telomeres using somatic cell hybridisation. Lorne Cancer Conference, Lorne, Vic., February 1998. Reddel, R.R., Colgin, L., Perrem, K.T., Dunham, M.A., Englezou, A., Bowtell, D.D.L. and Kilian, A. Telomere maintenance in telomerase-negative cell lines. Geron Symposium No. 2: Telomerase and Telomere Dynamics in Cancer and Aging, Maui, August 1998. Reddel, R.R., Bryan, T.M., Chang, A.C.-M., Colgin, L., Dalla-Pozza, L., Dunham, M.A., Englezou, A., Moy, E.L., Neumann, A.A., Noble, J.R. and Perrem, K.T. Genetic changes during immortalisation of human cells. American Association for Cancer Research Annual Scientific Meeting, New Orleans, March/April, 1998. Perrem K., Moy E., Bryan T., and Reddel R.R. Evidence for rapid shortening of telomeres in hybrids of telomerase positive X telomerase negative immortalised human cells. Lorne Cancer Conference, Lorne, Vic., February 1999. Reddel, R., Bonnefin, P., Colgin, L., Englezou, A., Perrem, K., Toouli, C. Telomere maintenance mechanisms and immortalisation of human cells. Conference on Human Cell Transformation, Cork, July 1999. Perrem K., Yeager T., and Reddel R.R. Telomere length dynamics in human somatic cell hybrids: evidence for a telomere length feedback control mechanism. Miami Nature Biotechnology Winter Symposium, Miami FL, USA, February 2000. Reddel, R., Colgin, L., Dunham, M., Englezou, A., Fasching, C., Neumann, A., Perrem, K. and Toouli, C. Telomere maintenance mechanisms in immortalised human cells. Lorne Cancer Conference, Lorne, Vic., February 2000. Perrem K., Colgin L.M,, Neumann A.A,, Yeager T. R., and Reddel R.R. Coexistence of ALT and telomerase. Lorne Cancer Conference, Lorne, Vic., February 2001. Perrem K., Colgin L.M,, Neumann A.A,, Yeager T. R., and Reddel R.R. Telomerase does not repress the ALT mechanism. ‘Telomeres and telomerase’, Cold Spring Harbor, NY, USA, March 2001. vi Summary Telomeres are specialised structures, consisting of TTAGGG DNA repeats and binding proteins, that cap the ends of human chromosomes and maintain chromosome integrity. It has been shown that telomeres shorten with each round of cell division in most normal human somatic cells. It has become generally accepted that this shortening is due, in part, to the inability of DNA polymerases to replicate the extreme ends of chromosomes which is a phenomenon known as the “end replication problem”. An intriguing hypothesis that has emerged from these observations is that critically shortened telomeres trigger growth arrest and senescence. This is regarded as a key determining factor in the limited lifespan of normal cells in culture and is commonly known as the “Telomere Hypothesis of Senescence”. In support of this hypothesis it has been demonstrated that immortalised human cells, that have an unlimited lifespan in culture, maintain stable telomere lengths and do not undergo progressive telomere shortening. In most cases this is due to the ribonucleoprotein enzyme telomerase, the activation of which is as a key step in the immortalisation process. Telomerase compensates for sequential telomere shortening by utilising an RNA template to catalyse the addition of repeat sequences by reverse transcription. It is absent from most normal tissue but is present in the germline and is presumably downregulated during development. Significantly, analysis of human tumour cells has shown that a majority also have active telomerase, which supports the importance of immortalisation in tumourigenesis. Previous work in this laboratory has shown that, although the majority of in vitro immortalised cells and tumour cells that have been studied maintain telomeres by reactivation of telomerase, a proportion do not have detectable telomerase activity. These telomerase-negative cells still maintain telomeres, however, and this is via a mechanism(s) yet to be fully elucidated known as Alternative Lengthening of Telomeres (ALT). ALT is characterised, in addition to lack of telomerase activity, by extreme telomere length heterogeneity with telomere lengths ranging from over 50 kilobases (kb) of DNA to almost undetectable. This phenotype is evident, by Southern analysis and fluorescent in situ hybridisation (FISH), in all ALT cells. Alternative mechanisms of telomere maintenance, via retrotransposition and recombination, had already been characterised in lower eukaryotes. It has been shown in this laboratory that ALT cell lines and tumours contain a novel type of PML body, referred to as ALT-associated PML bodies (APBs). APBs have been found in all of the ALT cell lines so far tested and also in archival tumour sections, and contain a number of factors which co-localise. These include PML, TTAGGG repeats, TRF 1 & TRF 2 telomere vii binding proteins and proteins involved in homologous recombination: RAD51 & RAD52. More recently, it has been shown that the RAD50/Mre11/Nbs1 complex, which is involved in cell cycle checkpoint control and repair of DNA damage, is also present in APBs. The presence of these RAD proteins in APBs is of great interest as a recombination between telomeres has been proposed as the central mechanism by which ALT lengthens telomeres.
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