Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform

© 2018 The Authors. Immortalising primary cells with hTERT has been common practice to enable primary cells to be of extended use in the laboratory since they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behaviour. Control and the premature ageing disease - Hutchinson-Gilford Progeria Syndrome primary dermal fibroblasts, with and without the classical G608G mutation have been immortalised with exogenous hTERT. However, hTERT immortalisation surprisingly elicits genome reorganisation, in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mis-localised in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalised cells and resulted in these mis-localised internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mis-localisation, which can be restored with a drug treatment that results in telomere re-shortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT immortalised cell lines.Brunel Progeria Research Fun

FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence

This is the author accepted manuscript. The final version is available from FASEB via the DOI in this recordCellular plasticity is a key facet of cellular homeostasis requiring correct temporal and spatial patterns of alternative splicing. Splicing factors, which orchestrate this process, demonstrate age-related dysregulation of expression; they are emerging as potential influences on aging and longevity. The upstream drivers of these alterations are still unclear but may involve aberrant cellular signaling. We compared the phosphorylation status of proteins in multiple signaling pathways in early and late passage human primary fibroblasts. We then assessed the impact of chemical inhibition or targeted knockdown of direct downstream targets of the ERK and AKT pathways on splicing factor expression, cellular senescence, and proliferation kinetics in senescent primary human fibroblasts. Components of the ERK and AKT signaling pathways demonstrated altered activation during cellular aging. Inhibition of AKT and ERK pathways led to up-regulation of splicing factor expression, reduction in senescent cell load, and partial reversal of multiple cellular senescence phenotypes in a dose-dependent manner. Furthermore, targeted knockdown of the genes encoding the downstream targets FOXO1 or ETV6 was sufficient to mimic these observations. Our results suggest that age-associated dysregulation of splicing factor expression and cellular senescence may derive in part from altered activity of ERK and AKT signaling and may act in part through the ETV6 and FOXO1 transcription factors. Targeting the activity of downstream effectors of ERK and AKT may therefore represent promising targets for future therapeutic intervention.-Latorre, E., Ostler, E. L., Faragher, R. G. A., Harries, L. W. FOXO1 and ETV6 genes may represent novel regulators of splicing factor expression in cellular senescence.This work was supported by The Dunhill Medical Trust [grant number: R386/1114]

Suppression of the senescence-associated secretory phenotype (SASP) in human fibroblasts using small molecule inhibitors of p38 MAP kinase and MK2.

Senescent cells show an altered secretome profile termed the senescence-associated secretory phenotype (SASP). There is an increasing body of evidence that suggests that the accumulation of SASP-positive senescent cells in humans is partially causal in the observed shift to a low-level pro-inflammatory state in aged individuals. This in turn suggests the SASP as a possible therapeutic target to ameliorate inflammatory conditions in the elderly, and thus a better understanding of the signalling pathways underlying the SASP are required. Prior studies using the early generation p38 MAPK inhibitor SB203580 indicated that p38 signalling was required for the SASP. In this study, we extend these observations using two next-generation p38 inhibitors (UR-13756 and BIRB 796) that have markedly improved selectivity and specificity compared to SB203580, to strengthen the evidence that the SASP is p38-dependent in human fibroblasts. BIRB 796 has an efficacy and toxicity profile that has allowed it to reach Phase III clinical trials, suggesting its possible use to suppress the SASP in vivo. We also demonstrate for the first time a requirement for signalling through the p38 downstream MK2 kinase in the regulation of the SASP using two MK2 inhibitors. Finally, we demonstrate that a commercially-available multiplex cytokine assay technology can be used to detect SASP components in the conditioned medium of cultured fibroblasts from both young and elderly donors. This assay is a high-throughput, multiplex microtitre-based assay system that is highly sensitive, with very low sample requirements, allowing it to be used for low-volume human biological fluids. Our initial studies using existing multiplex plates form the basis for a "SASP signature" assay that could be used as a high-throughput system in a clinical study setting. Our findings therefore provide important steps towards the study of, and intervention in, the SASP in human ageing and age-related disease

Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease

WRN is a RecQ helicase with an associated exonuclease activity important in DNA metabolism, including DNA replication, repair and recombination. In humans, deficiencies in WRN function cause the segmental progeroid Werner syndrome (WS), in which patients show premature onset of many hallmarks of normal human ageing. At the cellular level, WRN loss results in rapid replicative senescence, chromosomal instability and sensitivity to various DNA damaging agents including the topoisomerase inhibitor, camptothecin (CPT). Here, we investigate the potential of using either transient or stable WRN knockdown as a means of sensitising cells to CPT. We show that targeting WRN mRNA for degradation by either RNAi or hammerhead ribozyme catalysis renders human fibroblasts as sensitive to CPT as fibroblasts derived from WS patients, and furthermore, we find altered cell cycle transit and nucleolar destabilisation in these cells following CPT treatment. Such WS-like phenotypes are observed despite very limited decreases in total WRN protein, suggesting that levels of WRN protein are rate-limiting for the cellular response to camptothecin. These findings have major implications for development of anti-WRN agents that may be useful in sensitising tumour cells to clinically relevant topoisomerase inhibitors. © 2011 Springer Science+Business Media B.V

Small molecule modulation of splicing factor expression is associated with rescue from cellular senescence

Background: Altered expression of mRNA splicing factors occurs with ageing in vivo and is thought to be an ageing mechanism. The accumulation of senescent cells also occurs in vivo with advancing age and causes much degenerative age-related pathology. However, the relationship between these two processes is opaque. Accordingly we developed a novel panel of small molecules based on resveratrol, previously suggested to alter mRNA splicing, to determine whether altered splicing factor expression had potential to influence features of replicative senescence. Results: Treatment with resveralogues was associated with altered splicing factor expression and rescue of multiple features of senescence. This rescue was independent of cell cycle traverse and also independent of SIRT1, SASP modulation or senolysis. Under growth permissive conditions, cells demonstrating restored splicing factor expression also demonstrated increased telomere length, re-entered cell cycle and resumed proliferation. These phenomena were also influenced by ERK antagonists and agonists. Conclusions: This is the first demonstration that moderation of splicing factor levels is associated with reversal of cellular senescence in human primary fibroblasts. Small molecule modulators of such targets may therefore represent promising novel anti-degenerative therapies

Experimental flow studies in glaucoma drainage device development

AIMS—(I) To examine whether small holes produced by 248 nm excimer laser ablation in a polymer substrate could consistently produce a pressure drop in the desired target range (5-15 mm Hg) at physiological aqueous flow rates for use as an internal flow restrictor in a glaucoma drainage device, and (ii) to investigate whether external leakage could be reduced in comparison with conventional tube and plate glaucoma drainage devices by redesigning the exterior cross sectional shape of the portion contained within the sclerocorneal tunnel.
METHODS—Single holes with target diameters of 10 µm, 15 µm, 20 µm, and 25 µm were drilled using a 248 nm excimer laser in sample discs (n=6 at each diameter) punched from a 75 µm thick polyimide sheet. Sample discs were tested in a flow rig designed to measure the pressure drop across the discs. Using filtered, degassed water at a flow rate of 1.4 µl/min repeated flow measurements were taken (n=6) for each disc. After flow testing, all discs were imaged using a scanning electron microscope and the dimensions of each hole were derived using image analysis software. In the external leakage study, corneoscleral buttons (n=13) were prepared from cadaver pig eyes and mounted on an artificial anterior chamber infused with Tyrode solution. After the pressure had stabilised, standard occluded silicone tube implants were inserted through 23 gauge needle stab incisions at the limbus. These were compared against prototype PMMA implants with a novel shape profile inserted through 1.15 mm width microvitreoretinal (MVR) stab incisions at the limbus. The infusion rate was maintained and a second pressure measurement was taken when the pressure had stabilised. The difference between the first and second pressure measurement was then compared, as an index of external leakage.
RESULTS—Ablated tubes were found to have a near perfect circular outline on both the entry and exit side. The observed pressure drops across the ablated sample discs at each target diameter were as follows: 10 µm, mean 25.66 (SD 4.9) mm Hg; 15 µm, 6.7 (1.15); 20 µm, 1.66 (1.07); and 25 µm, <0.1 mm Hg. A strong correlation was observed between observed pressure drops and those predicted by Poiseuille's formula (R(2) =0.996). Target ablations of 15 µm diameter produced tubes that consistently achieved a pressure drop within the desired range (5-15 mm Hg). In the external leakage study, preinsertion pressures (mm Hg; mean (SD)) were 19.00 (4.3) (conventional method) and 20.00 (3.9) (new technique with PMMA prototypes). Post-insertion pressures were significantly reduced (10.40 (7.7); p<0.01) for the conventional technique and were essentially unchanged for the new technique (18.80 (4.9); p>0.1).
CONCLUSIONS—It was shown that it is possible, in principle, to control the dimensions of a manufactured tubular lumen in a glaucoma drainage device accurately enough to provide consistent protection from hypotony in the early period after glaucoma filtration surgery. By redesigning the external profile of glaucoma drainage device and incision technique, it was also shown that it is possible to eliminate uncontrolled external leakage.