Telomere length profiles in primary human peritoneal mesothelial cells are consistent with senescence

Mesothelial cell (MC) senescence contributes to malignancy and tissue fibrosis. The role of telomere erosion in MC senescence remains controversial, with evidence for both telomere-dependent and telomere-independent mechanisms reported. Single telomere length analysis revealed considerable telomere length heterogeneity in freshly isolated human peritoneal MCs, reflecting a heterogeneous proliferative history and providing high-resolution evidence for telomere-dependent senescence. By contrast the attenuated replicative lifespan, lack of telomere erosion and induction of p16 expression in in vitro-aged cells was consistent with stress-induced senescence. Given the potential pathophysiological impact of senescence in mesothelial tissues, high-resolution MC telomere length analysis may provide clinically useful information

T cell memory revisited using single telomere length analysis

The fundamental basis of T cell memory remains elusive. It is established that antigen stimulation drives clonal proliferation and differentiation, but the relationship between cellular phenotype, replicative history, and longevity, which is likely essential for durable memory, has proven difficult to elucidate. To address these issues, we used conventional markers of differentiation to identify and isolate various subsets of CD8+ memory T cells and measured telomere lengths in these phenotypically defined populations using the most sensitive technique developed to date, namely single telomere length analysis (STELA). Naive cells were excluded on the basis of dual expression of CCR7 and CD45RA. Memory subsets were sorted as CD27+CD45RA+, CD27intCD45RA+, CD27−CD45RA+, CD27+CD45RAint, CD27−CD45RAint, CD27+CD45RA−, and CD27−CD45RA− at >98% purity. The shortest median telomere lengths were detected among subsets that lacked expression of CD45RA, and the longest median telomere lengths were detected among subsets that expressed CD45RA. Longer median telomere lengths were also a feature of subsets that expressed CD27 in compartments defined by the absence or presence of CD45RA. Collectively, these data suggested a disconnect between replicative history and CD8+ memory T cell differentiation, which is classically thought to be a linear process that culminates with revertant expression of CD45RA

Cooperative Anti-Invasive Effect of Cdc42/Rac1 Activation and ROCK Inhibition in SW620 Colorectal Cancer Cells with Elevated Blebbing Activity

<div><p>Rho GTPases are key regulators of tumour cell invasion and therefore constitute attractive targets for the design of anticancer agents. Several strategies have been developed to modulate their increased activities during cancer progression. Interestingly, none of these approaches took into account the existence of the well-known antagonistic relationship between RhoA and Rac1. In this study, we first compared the invasiveness of a collection of colorectal cancer cell lines with their RhoA, Rac1 and Cdc42 activities. A marked decrease of active Cdc42 and Rac1 correlated with the high invasive potential of the cell lines established from metastatic sites of colorectal adenocarcinoma (LoVo, SKCo1, SW620 and CoLo205). Conversely, no correlation between RhoA activity and invasiveness was detected, whereas the activity of its kinase effector ROCK was higher in cancer cell lines with a more invasive phenotype. In addition, invasiveness in these colon cancer cell lines was correlated with a typical round and blebbing morphology. We then tested whether treatment with PDGF to restore Cdc42 and Rac1 activities and/or with Y27632, a chemical inhibitor of ROCK, could decrease the invasiveness of SW620 cells. The association of both treatments substantially decreased the invasive potential of SW620 cells and this effect was accompanied by loss of membrane blebbing, restoration of a more elongated cell morphology and re-establishment of E-cadherin-dependent adherens junctions. This study paves the road to the development of therapeutic strategies in which different Rho GTPase modulators are combined to modulate the cross-talk between Rho GTPases and their specific input in metastatic progression.</p> </div

PDGF and Y27632 treatments promote elongation of colon cancer cells.

<p>(a) Still DIC time-lapse images of cells treated or not (control) with PDGF and Y27632 (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s001" target="_blank">videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s005" target="_blank">S5</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344.s006" target="_blank">S6</a>). Frames show the change in morphology (from round to a more elongated shape) of the cells at the beginning of the movies. Scale bars, 10 µm. (b) DIC time-lapse images of SW620 cells before and after treatment with PDGF + Y27632 (from video S7). Frames show the change in cell morphology at the indicated times. Scale bars, 10 µm. (b) Quantification of cell elongation. A cell was considered elongated when its longest dimension was twice the shortest one and when it showed at least one protrusion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#pone.0048344-SanzMoreno1" target="_blank">[23]</a>. Histograms represent the percentage of elongated SW620 cells following treatment or not (control) with PDGF and/or Y27632 for 24 hours. Values are the mean +/− SD (error bars) of three independent experiments and the statistical significance was calculated using the unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001. (d) Cdc42 activity was determined following treatment or not (control) with PDGF for 24 hours as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#s4" target="_blank">Materials and Methods</a>. Values are the mean +/− SD (error bars) of four independent experiments and the statistical significance was calculated using the unpaired t-test. *p<0.05. (e) Rac1 activity was determined following treatment or not (control) with PDGF for 24 hours as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#s4" target="_blank">Materials and Methods</a>. Values are the mean +/− SD (error bars) of four independent experiments and the statistical significance was calculated using the unpaired t-test. *p<0.05. (f) SW620 cells were lysed and the amount of phosphorylated Myosin Light Chain 2 (MLC2) and of total MLC2 present in the lysates was determined by immunoblotting at different time-points (0 to 30 minutes) following treatment with 10 µM Y27632. Shown is a representative immunoblot.</p

PDGF and Y27632 impair cancer cell invasion.

<p>The invasiveness of SW620 colon cancer cells following treatment or not (control) with PDGF and/or Y27632 was quantified by using Matrigel invasion assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048344#s4" target="_blank">Materials and Methods</a>. Values are the mean +/− SD (error bars) of at least three independent experiments and the statistical significance was calculated using the unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001, ns non-significant.</p

The combined treatment with PDGF and Y27632 induces the re-establishment of E-cadherin junctions.

<p>(a) Representative E-cadherin staining in colon cancer cells treated or not (control) with PDGF and Y27632 for 24 hours. Scale bars, 10 µm. (b) Quantification of E-cadherin junctions. We scored as positive every cell showing at least one E-cadherin junction with one or more neighbouring cells. Histograms represent the percentage of SW620 colon cancer cells with E-cadherin junctions following treatment or not with PDGF and/or Y27632 for 24 hours. Values are the mean +/− SD (error bars) of three independent experiments and the statistical significance was calculated using the unpaired t-test. * p<0.05, ** p<0.01, *** p<0.001.</p

The oscillation hypothesis.

<p>Hypothetical illustration of RTL changes over time at the individual (solid line) and population (dotted line) level, based on the collected data from the present study and the literature.</p

XpYp telomere end distributions in single telomere length analysis (STELA) (6 lanes/sample).

<p>A. Comparison between blood donors 5 and 2 (control), showing similar patterns as the results from RT-PCR and Southern blot. The 3 month data for Donor 2 could not be included in the STELA, due to a lack of DNA after the Southern blot analysis. B. The telomere distribution in the samples from Donor 5, showing a considerable decrease in heterogeneity and a loss of the largest telomeres.</p