Physiological responses to genomic instability

Abnormalities in the cellular senescence programme may contribute to premature ageing and cancer predisposition. Primary human cells have a limited proliferative capacity, entering a state of irreversible growth arrest/senescence arising from telomere attrition (replicative senescence). Stress-induced premature senescence (SIPS) can additionally limit cellular lifespan and there are several human genomic instability (Gl) disorderscancer-prone/premature ageing syndromesin which SIPS may prematurely limit cellular lifespan. The hypothesis is that increased Gl is a critical factor in limiting the lifespan of somatic cells by activating common pathways leading to SIPS. This study examines the extent to which Gl cells present restraints on cellular proliferation with a focus on the degree to which they share similar properties not least a common dependence on the p38 mitogen-activated protein kinase (MAPK) mediated stress response pathway. The study utilised a panel of normal (N) and a Gl panel of fibroblasts comprising: Rothmund Thomson syndrome (RTS), Bloom syndrome (BS), Cockayne syndrome A (CSA), Hutchinson-Gilford Progeria syndrome (HGPS), Nijmegen Breakage syndrome (NBS), Werner syndrome (WS), Seckel syndrome (SS), Dyskeratosis Congenita (DKC) and Ligase 4 syndrome (LIG4). The study revealed a commonality of response to p38 inhibitors—increased clonogenicity and lifespan—for normal and Gl syndrome cells, suggesting that all cells undergo some degree of p38-mediated proliferation restraint/SIPS. A degree of inter-strain heterogeneity for reduced replicative potential and/or altered morphology was apparent for both normal and Gl syndrome primary fibroblasts. This study revealed truncated lifespans for LIG4, SS, HGPS, BS, RTS, DKC and NBS cells. GM18366(SS) and GM07166(NBS) cell strains show an altered morphology and increased p38 inhibitor responses suggesting elevated SIPS operation. Growth and DNA damage studies using a newly created bank of hTert-immortalised cell strains AG16409(N), AG18371(RTS), GM02548(BS), GM07166(NBS), GM01856(CSA), AG11498(HGPS) and GM17523(LIG4) showed that Gl syndrome cells, alleviated from the stress of telomere attrition, remain susceptible to SIPS. Of these RTS cells were hTert-immortalised for the first time, showing that senescence in these strains is telomere-dependent. Cell cycle analysis revealed a common p38- associated mechanism that limits retention in cycle. The specific p38 inhibitor BIRB796 was found to increase clonogenic potential of both normal and Gl syndrome cells. The findings suggest that both RTS and WS cells show a higher proportion of cells that are non-dividing. Furthermore, the study suggests that WS cells have an altered cell cycle transit time that is reduced by BIRB796 treatment. Overall, the study highlights successful single cell analysis approaches for testing the impact of p38 inhibitors by teasing out the nature of cellular heterogeneity arising from different sources for specific cell strains. These findings support and expand previous evidence that WS cells undergo higher levels of SIPS compared to normal cells and provide new evidence that RTS cells may also show an increased propensity for SIPS.