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Dissecting the cause of premature replicative senescence of Werner syndrome cells using selective chemical inhibitors

Reference	BB/D524140/1
Principal Investigator / Supervisor	Professor David Kipling
Co-Investigators / Co-Supervisors	Professor Mark Christopher Bagley, Dr Terence Davis
Institution	Cardiff University
Department	Cardiff University Department
Funding type	Research
Value (£)	420,603
Status	Completed
Type	Research Grant
Start date	27/02/2006
End date	26/06/2009
Duration	40 months

Abstract

Replicative senescence, the finite proliferative capacity of normal human cells in culture, could potentially contribute to the ageing of division-competent tissues. A powerful model system to study this is the genetic disease Werner¿s syndrome (WS). WS individuals age approximately 4-5 times faster than normal, but this dramatic premature ageing is largely restricted to division-competent tissues. Cultured WS fibroblasts undergo fewer population doublings (PDs) than their wild-type equivalents, providing a plausible explanation for the premature ageing phenotype of WS individuals. Both normal and WS fibroblasts use the progressive erosion of chromosomal telomeres as a cell division `counter¿, as shown by their immortalisation following forced expression of telomerase. However, our recent high-resolution analysis has argued against accelerated telomere erosion per cell division being responsible for their premature senescence. Rather, our data are consistent with an additional, telomere-independent mechanism operating in WS, one that synergises with telomere-driven senescence (TDS). Our recent work has provided a major clue as to its identity. Treatment of cultured WS cells with SB203580, an inhibitor of p38MAPK, essentially prevents all their premature ageing phenotypes. SB203580-treated WS cultures show a normal, `youthful¿ morphology, their growth rate reverts to wild-type rates, and their cellular lifespan (number of PD achieved) is extended to be within the range observed for normal fibroblasts. In contrast, the senescence of normal human fibroblasts is largely unaffected by SB203580. We speculate that the absence of the WRNp recQ helicase, which has been shown to cause stalled replication forks, results in a `replication stress¿ signal that is transduced by p38MAPK and synergises with TDS. Because this is a response to a fundamental defect in DNA replication, it is likely that a similar stress will also occur in vivo, in the proliferative tissues in WS individuals. Here we will use the power of chemical genetics to create compound libraries based on known p38 inhibitors that have different binding kinetics in order to probe and dissect the stress related signaling pathways involving p38. We will address the extent to which p38MAPK, as opposed to other proteins, is the relevant target of SB203480 in this system, and whether this pathway synergises with TDS via one of two potential common pathway components (p53 and p21Waf1). The major methods used will be: heterocyclic chemical syntheses, the culture of primary and immortalised WS fibroblasts, immunocytochemistry/Western blotting, and specific kinase assays. These will be used to design specific inhibitors and to monitor their effects on both the WS in vitro phenotype and to address specific questions (e.g. p53, HSP27 phosphorylation). The results from this study will assess the ability of a range of p38 inhibitors, with very different modes of binding, substrate selectivity and inhibitory potencies, to prevent premature cell ageing of WS fibroblasts in vitro. These experiments will test our hypothesis that stress signal transduction in WS cells is responsible for the accelerated replicative decline and is transduced by the stress-activated mitogen kinase p38alpha. The goal of this programme is to use chemical inhibitors to intervene in the rapid ageing of WS cells and in so doing understand the biological system that causes cell senescence and morphology changes. The outcome of this proposal will add to this knowledge and, it is hoped, will provide valuable new insights into WS, the ageing process as a whole and the influence of p38alpha stress signaling on the fundamental mechanisms of ageing.

Summary

unavailable

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Ageing
Research Priority	X – Research Priority information not available
Research Initiative	Selective Chemical Intervention In Biological Systems (SCIBS) [2005]
Funding Scheme	X – not Funded via a specific Funding Scheme

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