Telomere stability in cancer cells and the role of DNA mismatch repair
thesisposted on 15.12.2014, 10:38 by Aarón Méndez-Bermúdez
Human telomeres are essential for maintaining chromosome stability and are composed of TTAGGG repeat arrays with interspersed variant repeats at the proximal end. This work has focused on understanding the role of DNA mismatch repair on telomere stability by mapping the interspersion pattern of variant telomere repeats in search of mutations. Telomere instability was analysed in clones of mismatch repair (MMR) defective cell lines and colorectal carcinomas with MSH2 mutations. The combined mutation frequency found in four MMR-defective cell lines (3.6% per telomere per genome) and two colorectal tumours lacking the MSH2 gene (1.2% per telomere per genome) was significantly different compared to MMR proficient cell lines. The mutations resulted in losses, gains and telomere repeat-type changes suggesting that mutations originated by intra-allelic mechanisms. In addition, downregulation of the MSH2 gene was carried out in a normal fibroblast cell line using RNA interference. Downregulation of MSH2 caused microsatellite and telomere instability with a possible effect on telomere length. These results indicate that the MMR pathway is involved in maintaining the stability of telomeres. One variant telomere repeat found in humans is the CTAGGG repeat. When multiple tandem copies of CTAGGG are present in a telomere, it is highly unstable in the male germ-line with a mutation rate as high as 0.83 per sperm. In this work, the analysis of instability of CTAGGG-containing telomeres was extended in the germ-line and conducted in somatic tissue. The results indicated that the presence of more than five CTAGGG repeats cause instability in that telomere, with a mutation frequency of 3.8% per telomere per genome in somatic cells. Finally, the basis of the telomere maintenance mechanism (TMM) was investigated in a panel of liposarcomas where around 50% are reported to lack a TMM. By mapping telomeres from liposarcomas with unknown TMM, it was possible to identify complex mutations that arose, most probably, by recombination-like processes similar to that seen in the alternative lengthening of telomeres pathway (ALT). However, these tumours lack some of the markers for ALT such as ALT-associated PML bodies.