Heterogeneity in telomere length of human chromosomes

Vertebrate chromosomes terminate in variable numbers of T2AG3 nucleotide repeats. In order to study telomere repeats at individual chromosomes, we developed novel, quantitative fluorescence in situ hybridization procedures using labeled (C3TA2)3 peptide nucleic acid and digital imaging microscopy. Telomere fluorescence intensity values from metaphase chromosomes of cultured human hematopoietic cells decreased with the replication history of the cells, varied up to six- fold within a metaphase, and were similar between sister chromatid telomeres. Surprisingly, telomere fluorescence intensity values within normal adult bone marrow metaphases did not show a normal distribution, suggesting that a minimum number of repeats at each telomere is required and/or maintained during normal hematopoiesis.      

In vivo reduction of telomere length in human antigen-reactive memory T cells

There is a reduction in the average telomere lengths of CD4+ "memory" T cells, defined by the CD45RO+ phenotype, compared to CD54RA+ "naive" T cells. However, other studies suggest that telomerase activity often is sufficient to maintain the telomere length of certain B and T cell populations following immune activation in vivo. Thus it is uncertain whether genuine memory CD4+ T cells, defined by an immune response to specific recall antigens, would display telomeres of reduced length, or whether telomere size would be maintained. Therefore, we examined the telomere lengths of T cells responding to two common recall antigens, tetanus toxoid and Candida albicans. Telomere terminal restriction fragment length was assessed by Southern blots or by flow cytometry following in situ hybridization with telomere-specific peptide nucleic acid probes. For the five subjects tested, the Candida- or tetanus-reactive memory T cell populations demonstrated a significant reduction of telomere length even when compared to the phenotypically defined memory CD45RO+ T cell populations isolated from peripheral blood mononuclear cells. This finding suggests that telomerase activity does not fully compensate for the effects of in vivo activation and proliferation of some antigen-specific CD4+ T cell populations. This may contribute to immune senescence.     

The association of telomere length and genetic variation in telomere biology genesa

Telomeres cap chromosome ends and are critical for genomic stability. Many telomere‐associated proteins are important for telomere length maintenance. Recent genome‐wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in genes encoding telomere‐associated proteins (RTEL1 and TERT‐CLPTM1) as markers of cancer risk. We conducted an association study of telomere length and 743 SNPs in 43 telomere biology genes. Telomere length in peripheral blood DNA was determined by Q‐PCR in 3,646 participants from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial and Nurses' Health Study. We investigated associations by SNP, gene, and pathway (functional group). We found no associations between telomere length and SNPs in TERT‐CLPTM1L or RTEL1. Telomere length was not significantly associated with specific functional groups. Thirteen SNPs from four genes (MEN1, MRE11A, RECQL5, and TNKS) were significantly associated with telomere length. The strongest findings were in MEN1 (gene‐based P=0.006), menin, which associates with the telomerase promoter and may negatively regulate telomerase. This large association study did not find strong associations with telomere length. The combination of limited diversity and evolutionary conservation suggest that these genes may be under selective pressure. More work is needed to explore the role of genetic variants in telomere length regulation. Hum Mutat 31:1050–1058, 2010. Published 2010 Wiley‐Liss, Inc.     

Analyses and comparisons of telomerase activity and telomere length in human T and B cells: Insights for epidemiology of telomere maintenance

Telomeres are the DNA–protein complexes that protect the ends of eukaryotic chromosomes. The cellular enzyme telomerase counteracts telomere shortening by adding telomeric DNA. A growing body of literature links shorter telomere length and lower telomerase activity with various age-related diseases and earlier mortality. Thus, leukocyte telomere length (LTL) and telomerase activity are emerging both as biomarkers and contributing factors for age-related diseases. However, no clinical study has directly examined telomerase activity and telomere length in different lymphocyte subtypes isolated from the same donors, which could offer insight into the summary measure of leukocyte telomere maintenance.We report the first quantitative data in humans examining both levels of telomerase activity and telomere length in four lymphocyte subpopulations from the same donors—CD4+, CD8+CD28+ and CD8+CD28? T cells and B cells, as well as total PBMCs—in a cohort of healthy women. We found that B cells had the highest telomerase activity and longest telomere length; CD4+ T cells had slightly higher telomerase activity than CD8+CD28+ T cells, and similar telomere length. Consistent with earlier reports that CD8+CD28? T cells are replicatively senescent cells, they had the lowest telomerase activity and shortest telomere length. In addition, a higher percentage of CD8+CD28? T cells correlated with shorter total PBMC TL (r = ? 0.26, p = 0.05). Interestingly, telomerase activities of CD4+ and CD8+CD28+ T cells from the same individual were strongly correlated (r = 0.55, r < 0.001), indicating possible common mechanisms for telomerase activity regulation in these two cell subtypes. These data will facilitate the understanding of leukocyte aging and its relationship to human health.     

TRF1 is a critical trans-acting factor required for de novo telomere formation in human cells

The duplex telomere repeat (TTAGGG)(n) is an essential cis-acting element of the mammalian telomere, and an exogenous telomere repeat can induce chromosome breakage and de novo telomere formation at the site of a break (telomere seeding). Telomere seeding requires the telomere repeat (TTAGGG)(n) more stringently than does an in vitro telomerase assay, suggesting that it reflects the activity of a critical trans-acting element of the functional telomere, in addition to telomerase. Furthermore, telomere seeding is induced at a frequency fluctuating widely among human cell lines, suggesting variation in the activity of this hypothetical factor among cells. In this study, we investigated the cellular factor(s) required for telomere formation using the frequency of telomere seeding as an index and identified TRF1, one of the telomere repeat binding proteins, as an essential trans-acting factor. The exogenous telomere repeat induces telomere formation at a frequency determined by the availability of TRF1, even in telomerase-negative cells. Our study shows clearly that TRF1 has a novel physiological significance distinct from its role as a regulator of telomere length in the endogenous chromosome. The possible role of TRF1 in cell aging and immortalization is discussed.     

Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion

Human marrow stromal cells (MSCs) can be isolated from bone marrow and differentiate into multiple tissues in vitro and in vivo. These properties make them promising tools in cell and gene therapy. The lack of a specific MSC marker and the low frequency of MSCs in bone marrow necessitate their isolation by in vitro expansion prior to clinical use. This may severely reduce MSC proliferative capacity to the point that the residual proliferative potential is insufficient to maintain long-term tissue regeneration upon reinfusion. In this study we determined the effect of in vitro expansion on the replicative capacity of MSCs by correlating their rate of telomere loss during in vitro expansion with their behavior in vivo. We report that even protocols that involve minimal expansion induce a rapid aging of MSCs, with losses equivalent to about half their total replicative lifespan.     

The nature of human orgasm: a critical review of major trends

This critical review presents a synthesis of the available theoretical and empirical literatures on human orgasm. Findings from both normal and clinical human populations are included. Two major trends in the literature, the dichotomization of biological and psychological perspectives and the assumption of gender differences, are highlighted. A new multidimensional model of the psychological experience of orgasm is described with a view to futhering a biopsychological approach applicable to both sexes. Clinical applications of this new model are discussed.     

Measurement of telomere length in cells from pleural effusion: Asbestos exposure causes telomere shortening in pleural mesothelial cells

We estimated the telomere lengths of neoplastic and non‐neoplastic mesothelial cells and examined their correlation with asbestos exposure and the expression of markers of mesothelial malignancy. Cell blocks of pleural effusion obtained from 35 cases of non‐neoplastic disease (NN), 12 cases of malignant mesothelioma (MM) and 12 cases of carcinomatous effusion due to lung adenocarcinoma (LA) were examined. Fifteen of the 35 NN cases had pleural plaques (NNpp+) suggestive of asbestos exposure, and the other 20 cases had no pleural plaques (NNpp‐). Telomere length was measured using the tissue quantitative fluorescence in situ hybridization method, and expressed as normalized telomere‐to‐centromere ratio. NN cases had significantly longer telomeres than MM (P < 0.001) and LA (P < 0.001) cases. Telomeres in NNpp+ cases were slightly shorter than those of NNpp‐ cases (P = 0.047). MM and LA showed almost the same telomere length. NN cases with shorter telomeres tended to show aberrant expression of epithelial membrane antigen (EMA), CD146, glucose transporter 1 (GLUT1) and IGF‐II messenger RNA‐binding protein 3 (IMP3). These results suggest that telomere shortening and subsequent genetic instability play an important role in the development of MM. Measurement of telomere length of cells in pleural effusion might be helpful for earlier detection of MM.     

Telomerase and telomere length in normal and malignant human endometrium as prognostic markers

Telomerase activation and telomere length maintainence are thought to be essential for cellular immortality and oncogenesis. Normal human endometrium expresses significant telomerase activity in a menstrual phase dependent manner. In this report, we have evaluated telomerase activity and telomere length in post menopausal endometrial hyperplasias and endometrial cancers to study their usefulness as prognostic markers. Telomerase activity was measured by the TRAP assay (Boehringer Mannheim, Germany) and telomere restriction fragment (trf) by the telomere length assay kit (BD Pharmingen). Proliferation markers PCNA and Bcl2 were studied by immunohistochemistry. Senescence associated Ogal activity was studied simultaneously and correlated with the above markers. Strong telomerase activity was observed in the proliferative phase of the normal endometrium, endometrial cancers and post-menopausal endometrial hyperplasias compared to normal, secretory and resting phases of the endometrium. PCNA and Bcl2 showed high positivity in telomerase positive cases. Telomerase activity was inversely proportional to Ogal activity. Mean trf lengths became shortened as the normal tissues underwent neoplastic changes. Our study suggests that high telomerase activity and short telomere lengths could be useful prognostic markers in human endometrium.     

Interplay between telomere length and telomerase in human leukocyte differentiation and aging.

Blood leukocytes derive from bone marrow hematopoietic stem cells and differentiate into multiple types of mature cells that include granulocytes, monocytes, mast cells of myeloid lineage, and T and B lymphocytes of lymphoid lineage. Their distinctive paths of differentiation and unique roles in immune response provide a model for comparative analysis of biological parameters, such as telomere length and telomerase activity, in different types of leukocytes. Age has also been associated with the decline in immune functions and with the attrition of telomere length in leukocytes. This review will summarize recent progress in the study of telomere length and telomerase expression in leukocytes during differentiation and aging. In addition, I will attempt to shed new light on the roles of telomere and telomerase in leukocyte function and potential clinical interventions.     

Lack of correlation between telomere length and telomerase activity and expression in leukemic cells

The expression of three components of telomerase complex (hTR, hTERT, TP1) along with telomerase activity and telomere length in leukemic cells was investigated. Cells were isolated from peripheral blood and/or bone marrow of children with acute lymphoblastic (ALL) and non-lymphoblastic (ANLL) leukemia. Expression of three components of telomerase as well as telomerase activity was found in all leukemic cells. Chemiluminescent detection of terminal restriction fragments (TRF) from DNA isolated from ALL cells showed variable patterns expressing considerable heterogeneity of telomere length. The ALL cells appeared to have both long and short telomere lengths, in contrast to normal peripheral lymphocytes, which produced limited pattern of TRF. The ANLL cells produced predominantly short telomere pattern despite high telomerase activity and expression. It can be concluded that high telomerase activity and expression in leukemic cells is not always correlated with long telomeres (TRF pattern).     

Simultaneous flow cytometric analysis of cell surface markers and telomere length: analysis of human tonsilar B cells

Telomere Flow FISH is a recently developed method which allows the measurement of telomere length in purified subsets of cells using flow cytometry. However, the harsh conditions required for flow FISH have precluded its use with conventional cell surface staining, thus limiting its utility for large scale clinical studies. We have now developed a method which permits simultaneous analysis of cell surface markers along with telomere length estimation by flow cytometry. This new assay employs the covalent crosslinking of monoclonal antibodies conjugated with a heat stable fluorochrome to the cell surface prior to flow FISH. Using this technique we have confirmed that human germinal center B cells (IgD(-)/CD38(+)) have dramatically longer telomeres than pre-germinal center founder B cells (IgD(+)/CD38(+)). This approach simplifies the analysis of complex cell populations and will facilitate widespread investigation of telomere length in health and disease states.     

The effect of age and telomere length on immune function in the horse

Telomeres, specialized structures present at the ends of linear eukaryotic chromosomes, function to maintain chromosome stability and integrity. Telomeres shorten with each cell division eventually leading to replicative senescence, a process thought to be associated with age-related decline in immune function. We hypothesized that shortened PBMC telomere length is a factor contributing to immunosenescence of the aged horse. Telomere length was assessed in 19 horses ranging in age from 1 to 25 years. Mitogen-induced (3)H-thymidine incorporation, total serum IgG, and pro-inflammatory cytokine expression was also determined for each horse. Relative telomere length (RTL) was highly correlated with overall age. RTL was positively correlated with (3)H-thymidine incorporation and total IgG. Expression of pro-inflammatory cytokines was negatively correlated with RTL. These measures were also correlated with age, as expected. However, RTL was not correlated with immunosenescence and inflammaging in the oldest horse.     

Alcoholics show reduced telomere length in the oesophagus

Telomeres are repetitive G-rich DNA sequences located at the ends of chromosomes. Chromosomal and genomic instability due to telomere dysfunction plays an important role in carcinogenesis. To study telomere shortening in the oesophageal epithelium of alcoholics, we measured the telomere lengths of basal and parabasal cells in comparison with those of non-alcoholics using Q-FISH and our original software, Tissue Telo, and also assessed histological inflammation. Telomeres in basal cells were significantly shorter in alcoholics than in age-matched normal controls. Prominent histological findings of chronic inflammation were not evident in either alcoholics or non-alcoholics. Our finding that telomeres in the oesophageal epithelium are shorter in alcoholics than in non-alcoholics indicates that telomere shortening may be associated with the frequent occurrence of squamous cell carcinoma in alcoholics. Further studies to clarify the reason for the large annual loss of telomere length with rapid turnover or lower telomerase activity in the oesophageal epithelium of alcoholics will be necessary.     

Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients

The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells, and particularly HAART treated patients had shortened telomeres in all cell-subtypes. The finding that patients mobilized cells with an impaired proliferation to PWM during and after adrenalin infusion has possible clinical relevance for HIV infected patients during pathological stressful conditions, such as sepsis, surgery and burns. However, this study did not find a correlation between impaired proliferation and telomeres. It is concluded that physiological stress further aggravates the HIV-induced immune deficiency.     

Kinetics of DNA replication and telomerase reaction at a single-seeded telomere in human cells

In most cancer cells, telomerase is activated to elongate telomere DNA, thereby ensuring numerous rounds of cell divisions. It is thus important to understand how telomerase and the replication fork react with telomeres in human cells. However, the highly polymorphic and repetitive nature of the nucleotide sequences in human subtelomeric regions hampers the precise analysis of sequential events taking place at telomeres in S phase. Here, we have established HeLa cells harboring a single-seeded telomere abutted by a unique subtelomere DNA sequence, which has enabled us to specifically focus on the seeded telomere. We have also developed a modified chromatin immunoprecipitation (ChIP) method that uses restriction digestion instead of sonication to fragment chromatin DNA (RES-ChIP), and a method for immunoprecipitating 5-bromo-2'-deoxyuridine (BrdU)-labeled single-stranded DNA by incubating DNA with anti-BrdU antibody in the nondenaturing condition. We have shown that DNA replication of the seeded telomere takes place during a relatively narrow time window in S phase, and telomerase synthesizes telomere DNA after the replication. Moreover, we have demonstrated that the telomerase catalytic subunit TERT associates with telomeres before telomere DNA replication. These results provide a temporal and spatial framework for understanding DNA replication and telomerase reaction at human telomeres.     

The nature of virus-like inclusions in human red blood cells

Summary The author was unable to discover any difference in the erythrocytes of cancer patients and healthy persons by electron microscopic investigation.Hemoglobin granules and slight defects similar to the formations revealed by Reagan in erythrocytes of patients suffering fron virus diseases were observed in the erythrocytic stroma of both healthy persons and cancer patients.Presented by Active Member AMN SSSR N. N. Zhukov-Verezhnikov     

The role of telomere length modulation in delayed chromosome instability induced by ionizing radiation in human primary fibroblasts

Telomere integrity is important for chromosome stability. The main objective of our study was to investigate the relationship between telomere length modulation and mitotic chromosome segregation induced by ionizing radiation in human primary fibroblasts. We used X‐rays and low‐energy protons because of their ability to induce different telomeric responses. Samples irradiated with 4 Gy were fixed at different times up to 6 days from exposure and telomere length, anaphase abnormalities, and chromosome aberrations were analyzed. We observed that X‐rays induced telomere shortening in cells harvested at 96 hrs, whereas protons induced a significant increase in telomere length at short as well as at long harvesting times (24 and 96 hrs). Consistent with this, the analysis of anaphase bridges at 96 hrs showed a fourfold increase in X‐ray‐ compared with proton‐irradiated samples, suggesting a correlation between telomere length/dysfunction and chromosome missegregation. In line with these findings, the frequency of dicentrics and rings decreased with time for protons whereas it remained stable after X‐rays irradiation. Telomeric FISH staining on anaphases revealed a higher percentage of bridges with telomere signals in X‐ray‐treated samples than that observed after proton irradiation, thus suggesting that the aberrations observed after X‐ray irradiation originated from telomere attrition and consequent chromosome end‐to‐end fusion. This study shows that, beside an expected “early” chromosome instability induced shortly after irradiation, a delayed one occurs as a result of alterations in telomere metabolism and that this mechanism may play an important role in genomic stability. Environ. Mol. Mutagen. 54:172–179, 2013. © 2013 Wiley Periodicals, Inc.