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Title: Reversible inhibition of p16-mediated cellular senescence to alleviate ageing symptoms
Author: Castle, Joanna Toni
ISNI:       0000 0004 7427 1225
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2018
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The somatic mutation theory of ageing suggests that cell functional decline is a consequence of the accumulation of DNA mutations over an individual's lifetime. More recently ageing has been associated with increasing numbers of cells in the state of cell senescence, a loss of ability to divide following extensive cell division or genotoxic stresses. In particular, expression of the senescence effector protein p16 increases with age, and underlies a number of age-related illnesses. In engineered mouse models the specific removal of p16-positive cells halts certain ageing phenotypes. Chronic wounds are essentially non-healing and inflammatory wounds, primarily affecting aged individuals. They contain more senescent fibroblasts with upregulated p16 expression, compared to acute wounds. The aim of this project was to reverse senescence temporarily in aged normal human adult skin cells to allow proliferation and promote healing of chronic wounds. I designed four cell-penetrating peptides (CDK4P1-4), based on the p16-binding part of CDK4, aimed to bind p16 and prevent its growth-inhibitory action on CDK4. Their sequences all included Arg24 of CDK4, mutations of which abolish p16 binding. Neonatal human melanocytes show delayed senescence upon interference with pl6. However, using senescent adult melanocytes as a closer model for aged skin, I found that none of the peptides improved growth, indeed the two longer peptides (1 and 4) showed a concentration-dependent inhibition of growth with expression of cell-cycle inhibitor p21. CDK4P4 was used to study the mechanism of action further. It had both cellular and nuclear membrane penetrative ability and a half-life of around 24 hours. A negative control peptide was designed, with an Arg24Cys mutation to abolish pl6 binding. Interestingly, this peptide produced an increased inhibitory effect on adult melanocytes. CDK4P4 could also inhibit p16- deficient melanocytes. Thus the inhibition of growth did not require the presence of p16, while a positive growth component apparently did. This suggested that CDK4P4 had cyclin D as well as pl6 binding affinity, which was preliminarily supported by magnetic-pulldown immunoblotting. Further evidence for CDK4P4-p16 binding affinity was demonstrated by CDK4P4 treatment causing a phenotypic change from a flat, senescent to an elongated proliferative-like morphology of pl6-positive but not p16-negative senescent adult dermal fibroblasts. Growth was significantly and reproducibly increased with 10 pM CDK4P4 in both near-senescent and senescent fibroblasts. I have studied a series of peptides based on CDK4 with proven cellular and nuclear membrane penetration and apparent p16-binding ability, but often with counteracting inhibitory effects that may be mediated though cyclin D binding and/or increased p21 abundance. I have found some first circumstances in which the main study peptide CDK4P4 did increase division of the preferred target cells, senescent fibroblasts, and this provides a route for further study and development.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available