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 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. Studies in yeast have identified such a mechanism and it was proposed that a similar process occurred in human immortal cells that utilise ALT. It has now been shown by this laboratory that a recombination mechanism is indeed evident at the telomeres of ALT cells. To date all in vitro immortalised cell lines and most tumour cell types that have been studied have a telomere maintenance mechanism either via telomerase or ALT. Targeting telomerase has become a major focus of anti-cancer research as inhibitors have the potential to treat a wide variety of different tumour types. An understanding of ALT and its regulation is likely to be important in such therapeutic strategies, as selective pressure due to telomerase inhibition may result in ALT revertants within the tumour mass. Development of inhibitors of both telomerase and ALT may therefore be required when targeting telomere maintenance. The main focus of this thesis is the understanding of ALT repression in the SV40 immortalised skin fibroblast cell line GM847, as a means to further understanding the mechanism of ALT. The data presented provide new insights into the repression of ALT and also the relationship between telomerase and ALT which is important for our understanding of telomere maintenance in human cancer. Hybrids formed by fusion of normal cells and ALT cells underwent rapid telomere loss followed by senescence, indicating that normal cells contain factors that repress ALT. This strongly suggests that ALT is recessive and is activated in part by loss or mutation of repressors. Similar experiments were performed with ALT cells and telomerasepositive cells, and the resulting hybrids were all telomerase-positive and ALT repressed. It is possible that the same negative regulators are involved as additional data show that telomerase does not act as an ALT inhibitor. Exogenous expression of telomerase in ALT cells did not repress ALT, but both mechanisms co-existed in these transfected cells. This result provides a further argument for targeting both ALT and telomerase in any future treatments of tumours as it demonstrates in principle that these mechanisms are not mutually exclusive. A serendipitous finding was that a dominant-negative telomerase catalytic subunit caused telomere shortening in ALT cells, had not been reported elsewhere, and indeed was in contrast to previous findings. This provided further evidence for a link between telomerase and ALT as it suggested that there were essential components that were common to both pathways. As a further means to understanding ALT repression, a series of experiments was performed to determine the chromosomal localisation of ALT repressor(s) by microcell mediated chromosome transfer. This was done to facilitate the eventual isolation of repressors. A repressor of ALT in the chemically immortalised fibroblast cell line SUSM-1, had been reported to be localised to chromosome 7. This result could not be repeated in the GM847 cell line, but ALT repression was evident in GM847 cells upon transfer of chromosome 6. Another important question regarding the nature of ALT is the structure and sequence of the long heterogeneous telomeres generated by ALT specific recombination, which is the focus of the final series of data that is presented. ALT telomere length heterogeneity was detected under denaturing conditions, ruling out the possibility that it was an artefact of electrophoresis conditions due to novel secondary structure. Although the hybridisation signal intensity of TTAGGG increases at the onset of immortalisation in ALT cells, it had been demonstrated by restriction digests that degenerate repeats did exist at the telomeres of some ALT cell lines. Sequences containing telomere repeats were cloned from the ALT cell line WI38 VA13/2RA (SV40 immortalised fibroblasts) and these were found to be interspersed with a number of other sequence fragments. The significance of these sequences in relation to the mechanism of ALT is discussed.
| Identifer | oai:union.ndltd.org:ADTP/283151 |
| Date | January 2001 |
| Creators | Perrem, Kilian Thomas |
| Publisher | University of Sydney. Children's Medical Research Institute |
| Source Sets | Australiasian Digital Theses Program |
| Language | English, en_AU |
| Detected Language | English |
| Rights | Copyright Perrem, Kilian Thomas;http://www.library.usyd.edu.au/copyright.html |
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