My PhD thesis involves the experimental analysis of the mechanisms of telomere loss during the onset of replicative senescence, the potential nature of the telomere signal that triggers replicative senescence, and the possible contribution of telomere dynamics to aging and chromosome instability. Human telomeres are composed of the DNA sequence TTAGGG reiterated into arrays of upto 20kb, which together with associated proteins cap the ends of human chromosomes. The loss of telomeric sequences with ongoing cell division due to the end replication problem, ultimately results in the loss of telomeric function and the triggering of replicative senescence in many human cell types. Telomere erosion can therefore impose a limit on the replicative potential of human cell. This raises the possibility of a telomere-dependant accumulation of senescent cells in human tissue, which may underline age-related tissue deterioration, disease and cancer predisposition.
I have been investigating telomere dynamics using the Single Telomere Length Analysis (STELA), a high resolution technique that determines telomere length from single DNA molecules at specific chromosome ends. I have extended the orginal observations at the XpYp telomere by extending STELA analysis in include the telomeres of 17p, 11q, 12q, and 2p. Analysis of fibroblast populations passaged to senescence revealed two mechanisms that result in telomere loss; gradual erosion as a consequence of end-replication losses, and large-scale length changes termed human Telomere Rapid Deletion (hTRD). With the exception of 17p, the telomere dynamics were conserved amongst the telomeres analysed. However, the 17p telomere was more stable with a striking paucity of hTRD and displayed a trend towards being the shortest telomere, indeed this end exhibited the shortest recorded telomere length distribution (300bp) in senescent cells.. The mutational mechanisms that underlie hTRD is unclear. However it appears likely from genetic data in human cells ans observations in yeat that hTRD may involved intrachromatidal processes such as recombination with the telomere loop sgtructure, oxidative damage and sister chromatid exchange. The mechanisms underlying these mutational events are currently under investigation.
The ectopic over expression by the retrovirall transduction of the telomerase catalytic subunit (hTERT), resulted in the homogenisation of both allelic telomere length distributions and chromosome specific length distributions. However analysis of cancer derived keratinocytes expressing physiological levels of hTERT, revealed the maintenance of allelic distributions and chromosome specific lengths, where 17p was again the shortest analysed telomere. This phenomenon has also been shown in the male germ line, where 17p was consistently the shorter telomere, compared to XpYp. Such results suggest the presence of cis acting determinates of chromosome and indeed allele specific telomere lengths, that operate in the presence of telomerase.
Winning the best poster prize at the BSRA meeting in Oxford was an immense privilege, especially due to the quality of the other entrants. Winning this prize has boosted my confidence concerning presenting my work for public viewing, and has reassured me that my work is of reasonable quality. Winning the prize has also increased my incentive to work harder, and to improve the standard of presenting my work at other scientific meetings. I would like to thank the BSRA for the travel grant, and the thought provoking well organised 2006 meeting. I would also like to thank my amazing PhD supervisor, Duncan Baird for all his support and guidance.