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

CCR8 Expression Defines Tissue-Resident Memory T Cells in Human Skin

Human skin harbors two major T cell compartments of equal size that are distinguished by expression of the chemokine receptor CCR8. In vitro studies have demonstrated that CCR8 expression is regulated by TCR engagement and the skin tissue microenvironment. To extend these observations, we examined the relationship between CCR8+ and CCR8− skin T cells in vivo. Phenotypic, functional, and transcriptomic analyses revealed that CCR8+ skin T cells bear all the hallmarks of resident memory T cells, including homeostatic proliferation in response to IL-7 and IL-15, surface expression of tissue localization (CD103) and retention (CD69) markers, low levels of inhibitory receptors (programmed cell death protein 1, Tim-3, LAG-3), and a lack of senescence markers (CD57, killer cell lectin-like receptor subfamily G member 1). In contrast, CCR8− skin T cells are heterogeneous and comprise variable numbers of exhausted (programmed cell death protein 1+), senescent (CD57+, killer cell lectin-like receptor subfamily G member 1+), and effector (T-bethi, Eomeshi) T cells. Importantly, conventional and high-throughput sequencing of expressed TCR β-chain (TRB) gene rearrangements showed that these CCR8-defined populations are clonotypically distinct, suggesting unique ontogenies in response to separate antigenic challenges and/or stimulatory conditions. Moreover, CCR8+ and CCR8− skin T cells were phenotypically stable in vitro and displayed similar levels of telomere erosion, further supporting the likelihood of a nonlinear differentiation pathway. On the basis of these results, we propose that long-lived memory T cells in human skin can be defined by the expression of CCR8

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

Cofilin phosphorylation increases in the most invasive colon cancer cell lines.

<p>Cells were lysed and the amount of phosphorylated Cofilin and of total Cofilin present in the lysates was analysed by immunoblotting. (a) Representative immunoblot. (b) Quantification of Cofilin phosphorylation. Histograms represent the ratios of the phospho-Cofilin signal over the total Cofilin signal. Values are the mean +/− SD (error bars) of three independent experiments.</p

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

Extensive telomere erosion in the initiation of colorectal adenomas and its association with chromosomal instability

Background. Telomere shortening, dysfunction, and fusion may facilitate the acquisition of large-scale genomic rearrangements, driving clonal evolution and tumor progression. The relative contribution that telomere dysfunction and/or APC mutation play in the chromosome instability that occurs during colorectal tumorigenesis is not clear. Methods. We used high-resolution telomere length and fusion analysis to analyze 85 adenomatous colorectal polyps obtained from 10 patients with familial adenomatous polyposis and a panel of 50 colorectal carcinomas with patient-matched normal colonic mucosa. Telomerase activity was determined using the telomeric repeat amplification protocol. Array-CGH was used to detect large-scale genomic rearrangements. Pearson correlation and Student t test were used, and all statistical tests were two-sided. Results. Despite the presence of telomerase activity, we observed apparent telomere shortening in colorectal polyps that correlated with large-scale genomic rearrangements (P < .0001) but was independent of polyp size and indistinguishable from that observed in colorectal carcinomas (P = .82). We also observed apparent lengthening of telomeres in both polyps and carcinomas. The extensive differences in mean telomere length of up to 4.6kb between patient-matched normal mucosa and polyps were too large to be accounted for by replicative telomere erosion alone. Telomere fusion events were detected in both polyps and carcinomas; the mutational spectrum accompanying fusion was consistent with alternative nonhomologous end joining. Conclusions. Telomere length distributions observed in colorectal polyps reflect the telomere length composition of the normal originating cells from which clonal growth was initiated. Originating cells containing both short telomeres and APC mutations may give rise to polyps that exhibit short telomeres and are prone to telomere dysfunction, driving genomic instability and progression to malignancy

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