Investigating the reversibility of senescence

Senescence is defined as a permanent and irreversible cell cycle arrest. The initiating stimuli can be diverse, such as oncogene expression, reactive oxygen species and critically short telomeres. Irrespective of the nature of the trigger, p53-dependent DNA damage response (DDR) is initiated, leading to cell cycle arrest in G1. A prolonged DDR activation can further reinforce the arrest through the activation of the cyclin-dependent kinase inhibitor p16. The inability of the semi-conservative DNA replication machinery to completely copy the ends of linear eukaryotic chromosomes results in a gradual telomere shortening that is counteracted by telomerase, a ribonucleoprotein which can add telomeric repeats to the 3’ end of a chromosome. However, during human embryonic development, telomerase is downregulated through decreased expression of the telomerase catalytic subunit hTERT. Consequently, human somatic cells undergo progressive telomere shortening that will ultimately lead to eroded telomeres being recognised by the DDR and induction of replicative senescence. Reconstitution of hTERT in several human somatic cells has been shown to prevent replicative senescence. During cancer development, cell immortalisation requires escaping senescence and telomerase reactivation is the prevalent route to this end. However, while it is clear that the presence of telomerase can counteract entry into the senescence, it is not known if, once senescence has been established, telomerase could drive cells out of the arrest. In this work, I address the reversibility of senescence triggered by critically short telomeres by: 1) reactivating telomerase expression in senescent cells; 2) reactivating expression and tethering telomerase to telomeres during senescence; and 3) reactivating telomerase expression together with transient DDR inhibition. By taking advantage of telomerase fused to a conditional degron, I have tested if telomerase stabilisation in senescent MRC5 human fibroblasts could bypass the arrest. My results show that the presence of telomerase was not sufficient to overcome senescence. Since it has been shown that yeast telomerase can only gain access to telomeres during DNA replication, one possibility is that telomerase reactivated in senescent cells was unable to localise to telomeres. To address this possibility, I have expressed an hPOT1-hTERT fusion to tether telomerase to telomeres irrespective of the cell cycle stage. In a parallel approach, I have set up a system to allow senescent cells to enter the S-phase by transient inhibition of DDR, by IPTG-inducible knock-down of p53 and p16. This study contributes to improving our understanding of molecular mechanisms of senescence and its reversibility as a possible approach for the therapy of agerelated diseases