Telomere deprotection and the maintenance of genome integrity: discrepancy between telomere shortening and shelterin dysfunction
- Autores: Aina Bernal Martínez
- Directores de la Tesis: Laura Tusell Padrós (dir. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2019
- Idioma: español
- Tribunal Calificador de la Tesis: Neus Agell Jané (presid.), Judit Pampalona Sala (secret.), Giusy Tornillo (voc.)
- Programa de doctorado: Programa de Doctorado en Biología Celular por la Universidad Autónoma de Barcelona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TESEO
- Resumen
Telomeres are nucleoprotein structures that cap the end of chromosomes and protect them from illegitimate recombination through a lariat conformation or t-loop that is mainly promoted by TRF2 protein. Dysfunctional telomeres have been proved to be a mechanism capable of originating chromosome instability (CIN) in mouse and human cells, and promote tumorigenesis in mouse models.
This dissertation thesis aims to generate immortalised but unstable cells due to telomere deprotection through progressive telomere shortening and by TRF2 depletion, and to evaluate their tumorigenic potential. In Work I, p16INK4a-deficient human mammary epithelial cells (vHMECs) lacking or not for p53 function through specific short-hairpin RNA inactivation, were karyotyped at different population doublings to evaluate chromosomal abnormalities and their evolution. In the absence of telomerase, vHMECs progressively shortened their telomeres and subsequent end-to-end fusions initiated breakage-fusion-bridge (BFB) cycles and promoted CIN. However, these unstable cells finally succumbed to cell cycle arrest, independently of their p53 checkpoint status. In contrast, hTERT overexpression in p53-proficient vHMECs resulted in cells able to proliferate indefinitely with a nearly stable karyotype, while immortalised p53-deficient cells showed signs of CIN that could be permissive with an evolving karyotype.
In Work II and Work III, acute telomere deprotection was induced by t-loop disassembly through transient expression of the dominant negative form of TRF2 (TRF2ΔBΔM) in the mammary cell line MCF-10A and in immortalised HMEC derived from cosmetic reductions of four healthy donors, respectively. In Work II, acute telomere deprotection phenotype was reflected by the presence of TIFs and by an increase of end-to-end fusions and anaphase bridges after TRF2ΔBΔM induction. Anaphase bridges are considered the prelude to breakage-fusion-bridge cycles and subsequent karyotype reorganisations. However, no scars of BFB cycles or highly reorganised cells have been observed after transient expression of TRF2ΔBΔM, independently of the status of p53 and pRb proteins. Instead, diploid cells were enriched after successive cycles of telomere deprotection induction, thus suggesting that excessive telomere deprotection could be detrimental for the origin of cells with highly reorganised karyotypes. According with these results, in Work III, immortalised HMEC through hTERT and SV40LT overexpression transiently expressing TRF2ΔBΔM (HMEC-TO) exhibited an increase of anaphase bridges. But after a minimum of five cycles of telomere protection and deprotection, TRF2ΔBΔM expressing cells did not display scars of telomere deprotection and ongoing BFB cycles. In contrast to MCF-10A derived cell lines, the HMEC-TO cell lines exhibited a progressive increase of polyploid cells as a consequence of SV40LT immortalisation process. Independently of the cause of polyploidy increase, cells exposed to TRF2ΔBΔM expression cycles did not exhibit a telomere dysfunction phenotype or either a tumorigenic potential, thus suggesting that TRF2ΔBΔM expression provoked a deleterious effect over TRF2ΔBΔM expressing cells and prevented CIN emergence.
In conclusion, the present dissertation provides evidence that telomere dysfunction acts as a double sword mechanism for genome integrity. On the one hand, telomere shortening induces a mild and progressive DNA damage that firstly is compatible with cell viability, until damage is high enough to induce cell death. On the contrary, shelterin dysfunction affects widely to all chromosomes inducing an exacerbated cell response that is deleterious for cell viability and karyotype reorganisation. This Thesis illustrate that acute telomere deprotection through shelterin dysfunction could be a useful tool to impinge an exacerbated DNA damage and maintain genome integrity in human mammary cells.
