Measuring vertebrate telomeres: applications and limitations

Telomeres are short tandem repeated sequences of DNA found at the ends of eukaryotic chromosomes that function in stabilizing chromosomal end integrity. In vivo studies of somatic tissue of mammals and birds have shown a correlation between telomere length and organismal age within species, and correlations between telomere shortening rate and lifespan among species. This result presents the tantalizing possibility that telomere length could be used to provide much needed information on age, ageing and survival in natural populations where longitudinal studies are lacking. Here we review methods available for measuring telomere length and discuss the potential uses and limitations of telomeres as age and ageing estimators in the fields of vertebrate ecology, evolution and conservation

Lung fibroblasts from patients with emphysema show markers of senescence in vitro

BACKGROUND: The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD. METHODS: Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated β-galactosidase (SA-β-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant. RESULTS: The median (quartiles) percentage of fibroblasts positive for SA-β-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor ≥ 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema. CONCLUSION: These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate

Satellite Cells Senescence in Limb Muscle of Severe Patients with COPD

Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec, Québec, Canada Rationale: The maintenance of peripheral muscle mass may be compromised in chronic obstructive pulmonary disease (COPD) due to premature cellular senescence and exhaustion of the regenerative potential of the muscles. Methods: Vastus lateralis biopsies were obtained from patients with COPD (n = 16) and healthy subjects (n = 7). Satellite cell number and the proportion of central nuclei, as a marker of muscle regenerative events, were assessed on cryosections. Telomere lengths, used as a marker of cellular senescence, were determined using Southern blot analyses. Results: Central nuclei proportion was significantly higher in patients with COPD with a preserved muscle mass compared to controls and patients with COPD with muscle atrophy (p,0.001). In COPD, maximal telomere length was significantly decreased compared to controls (p,0.05). Similarly, minimal telomere length was significantly reduced in GOLD III–IV patients with muscle atrophy compared to controls (p,0.005). Minimal, mean and maximum telomere lengths correlated with mid-thigh muscle cross-sectional area (MTCSA) (R = 0.523, p = 0.005; R = 0.435, p = 0.019 and R = 0.491, p = 0.009, respectively). Conclusions: Evidence of increased regenerative events was seen in GOLD III–IV patients with preserved muscle mass. Shortening of telomeres in GOLD III–IV patients with muscle atrophy is consistent with an increased number of senescen

Sirt1 Deficiency Attenuates Spermatogenesis and Germ Cell Function

In mammals, Sirt1, a member of the sirtuin family of proteins, functions as a nicotinamide adenine dinucleotide-dependent protein deactylase, and has important physiological roles, including the regulation of glucose metabolism, cell survival, and mitochondrial respiration. The initial investigations of Sirt1 deficient mice have revealed a phenotype that includes a reduced lifespan, small size, and an increased frequency of abnormal sperm. We have now performed a detailed analysis of the molecular and functional effects of Sirt1 deficiency in the germ line of Sirt1 knock-out (−/−) mice. We find that Sirt1 deficiency markedly attenuates spermatogenesis, but not oogenesis. Numbers of mature sperm and spermatogenic precursors, as early as d15.5 of development, are significantly reduced (∼2-10-fold less; P≤0.004) in numbers in Sirt1−/− mice, whereas Sirt1 deficiency did not effect the efficiency oocyte production following superovulation of female mice. Furthermore, the proportion of mature sperm with elevated DNA damage (∼7.5% of total epididymal sperm; P = 0.02) was significantly increased in adult Sirt1−/− males. Analysis of global gene expression by microarray analysis in Sirt1 deficient testis revealed dysregulated expression of 85 genes, which were enriched (P<0.05) for genes involved in spermatogenesis and protein sumoylation. To assess the function of Sirt1 deficient germ cells, we compared the efficiency of generating embryos and viable offspring in in vitro fertilization (IVF) experiments using gametes from Sirt1−/− and sibling Sirt1+/− mice. While viable animals were derived in both Sirt1−/− X wild type and Sirt1−/− X Sirt1−/− crosses, the efficiency of producing both 2-cell zygotes and viable offspring was diminished when IVF was performed with Sirt1−/− sperm and/or oocytes. Together, these data support an important role for Sirt1 in spermatogenesis, including spermatogenic stem cells, as well as germ cell function

Endothelial and Smooth Muscle Cells from Abdominal Aortic Aneurysm Have Increased Oxidative Stress and Telomere Attrition

Background: Abdominal aortic aneurysm (AAA) is a complex multi-factorial disease with life-threatening complications. AAA is typically asymptomatic and its rupture is associated with high mortality rate. Both environmental and genetic risk factors are involved in AAA pathogenesis. Aim of this study was to investigate telomere length (TL) and oxidative DNA damage in paired blood lymphocytes, aortic endothelial cells (EC), vascular smooth muscle cells (VSMC), and epidermal cells from patients with AAA in comparison with matched controls. Methods: TL was assessed using a modification of quantitative (Q)-FISH in combination with immunofluorescence for CD31 or α-smooth muscle actin to detect EC and VSMC, respectively. Oxidative DNA damage was investigated by immunofluorescence staining for 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG). Results and Conclusions: Telomeres were found to be significantly shortened in EC, VSMC, keratinocytes and blood lymphocytes from AAA patients compared to matched controls. 8-oxo-dG immunoreactivity, indicative of oxidative DNA damage, was detected at higher levels in all of the above cell types from AAA patients compared to matched controls. Increased DNA double strand breaks were detected in AAA patients vs controls by nuclear staining for γ-H2AX histone. There was statistically significant inverse correlation between TL and accumulation of oxidative DNA damage in blood lymphocytes from AAA patients. This study shows for the first time that EC and VSMC from AAA have shortened telomeres and oxidative DNA damage. Similar findings were obtained with circulating lymphocytes and keratinocytes, indicating the systemic nature of the disease. Potential translational implications of these findings are discussed. © 2012 Cafueri et al

Troglitazone suppresses telomerase activity independently of PPARγ in estrogen-receptor negative breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Breast cancer is one the highest causes of female cancer death worldwide. Many standard chemotherapeutic agents currently used to treat breast cancer are relatively non-specific and act on all rapidly dividing cells. In recent years, more specific targeted therapies have been introduced. It is known that telomerase is active in over 90% of breast cancer tumors but inactive in adjacent normal tissues. The prevalence of active telomerase in breast cancer patients makes telomerase an attractive therapeutic target. Recent evidence suggests that telomerase activity can be suppressed by peroxisome proliferator activated receptor gamma (PPARγ). However, its effect on telomerase regulation in breast cancer has not been investigated.</p> <p>Methods</p> <p>In this study, we investigated the effect of the PPARγ ligand, troglitazone, on telomerase activity in the MDA-MB-231 breast cancer cell line. Real time RT-PCR and telomerase activity assays were used to evaluate the effect of troglitazone. MDA-MB-231 cells had PPARγ expression silenced using shRNA interference.</p> <p>Results</p> <p>We demonstrated that troglitazone reduced the mRNA expression of hTERT and telomerase activity in the MDA-MB-231 breast cancer cell line. Troglitazone reduced telomerase activity even in the absence of PPARγ. In agreement with this result, we found no correlation between PPARγ and hTERT mRNA transcript levels in breast cancer patients. Statistical significance was determined using Pearson correlation and the paired Student's <it>t </it>test.</p> <p>Conclusions</p> <p>To our knowledge, this is the first time that the effect of troglitazone on telomerase activity in breast cancer cells has been investigated. Our data suggest that troglitazone may be used as an anti-telomerase agent; however, the mechanism underlying this inhibitory effect remains to be determined.</p

Shortened Telomere Length Is Associated with Increased Risk of Cancer: A Meta-Analysis

BACKGROUND: Telomeres play a key role in the maintenance of chromosome integrity and stability, and telomere shortening is involved in initiation and progression of malignancies. A series of epidemiological studies have examined the association between shortened telomeres and risk of cancers, but the findings remain conflicting. METHODS: A dataset composed of 11,255 cases and 13,101 controls from 21 publications was included in a meta-analysis to evaluate the association between overall cancer risk or cancer-specific risk and the relative telomere length. Heterogeneity among studies and their publication bias were further assessed by the χ(2)-based Q statistic test and Egger's test, respectively. RESULTS: The results showed that shorter telomeres were significantly associated with cancer risk (OR = 1.35, 95% CI = 1.14-1.60), compared with longer telomeres. In the stratified analysis by tumor type, the association remained significant in subgroups of bladder cancer (OR = 1.84, 95% CI = 1.38-2.44), lung cancer (OR = 2.39, 95% CI = 1.18-4.88), smoking-related cancers (OR = 2.25, 95% CI = 1.83-2.78), cancers in the digestive system (OR = 1.69, 95% CI = 1.53-1.87) and the urogenital system (OR = 1.73, 95% CI = 1.12-2.67). Furthermore, the results also indicated that the association between the relative telomere length and overall cancer risk was statistically significant in studies of Caucasian subjects, Asian subjects, retrospective designs, hospital-based controls and smaller sample sizes. Funnel plot and Egger's test suggested that there was no publication bias in the current meta-analysis (P = 0.532). CONCLUSIONS: The results of this meta-analysis suggest that the presence of shortened telomeres may be a marker for susceptibility to human cancer, but single larger, well-design prospective studies are warranted to confirm these findings