Rovillain, E.; (2011) Dissecting the telomere-independent pathways underlying human cellular senescence. Doctoral thesis , UCL (University College London).

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Abstract

Cellular senescence is an irreversible program of cell cycle arrest triggered in normal somatic cells in response to a variety of intrinsic and extrinsic stimuli including telomere attrition, DNA damage, physiological stress and oncogene activation. Finding that inactivation of the pRB and p53 pathways by SV40-LT antigen cooperates with hTERT to immortalize cells has allowed us to use a thermolabile mutant of SV40-LT to develop human fibroblasts where the cells are immortal if grown at 34oC but undergo an irreversible growth arrest within 5 days at 38oC. When these cells cease dividing, senescence-associated-β-galactosidase (SA-β-Gal) activity is induced and the growth-arrested cells have many features of senescent cells. Since these cells growth-arrest in a synchronous manner, I have used Affymetrix expression profiling to identify the genes differentially expressed upon senescence. This identified 816 up- and 961 down-regulated genes whose expression was reversed when growth arrest was abrogated. I have shown that senescence was associated with activation of the NF-κB pathway and up-regulation of a number of senescence-associated-secretory-proteins including IL6. Perturbation of NF-κB signalling either by direct silencing of NF-κB subunits or by upstream modulation overcame growth-arrest indicating that activation of NF-κB signalling has a causal role in promoting senescence. I also applied a retroviral shRNA screen covering ~10,000 genes to the same cell model. Overlapping with the microarray data revealed particularly interesting targets, such as LTBP3 and Layilin. Finally, I profiled micro-rna expression. 15 of the top micro-rnas down-regulated upon senescence were chosen to express in the HMF3A system. 6 of them were able to bypass the growth-arrest. In conclusion, my work has uncovered novel markers involved in senescence as well as identifying that both activation of p53 and pRb pathway result in activation of NF-κB signalling which promotes senescence. Both results lead to a better understanding of senescence and its pathways.

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