DNA Telomere Length Research Articles

Monocyte Telomere Shortening and Oxidative DNA Damage in Type 2 Diabetes

Telomeres are DNA sequences necessary for DNA replication, which shorten at cell division at a rate related to levels of oxidative stress. Once shortened to a critical length, cells are triggered into replicative senescence. Type 2 diabetes is associated with oxidative DNA damage, and we hypothesized that telomere shortening would characterize type 2 diabetes. We studied 21 male type 2 diabetic subjects (mean age 61.2 years, mean HbA(1c) 7.9%) selected to limit confounding effects on telomere length and 29 matched control subjects. Telomere length was measured in peripheral venous monocyte and T-cells (naïve and memory) by fluorescent in situ hybridization and oxidative DNA damage by flow cytometry of oxidized DNA bases. Peripheral insulin resistance (homeostasis model assessment) and high-sensitivity C-reactive protein (hsCRP) were measured. Mean monocyte telomere length in the diabetic group was highly significantly lower than in control subjects (4.0 [1.1] vs. 5.5 [1.1]; P < 0.0001), without significant differences in lymphocyte telomere length. There was a trend toward increased oxidative DNA damage in all diabetes cell types examined and a significant inverse relationship between oxidative DNA damage and telomere length (r = -0.55; P = 0.018) in the diabetic group. Telomere length was unrelated to plasma CRP concentration or insulin resistance. Monocyte telomere shortening in type 2 diabetes could be due to increased oxidative DNA damage to monocyte precursors during cell division. This data suggests that monocytes adhering to vascular endothelium and entering the vessel wall in type 2 diabetes are from a population with shorter telomeres and at increased risk of replicative senescence within vascular plaque.

Mutations in Mre11 Phosphoesterase Motif I That Impair Saccharomyces cerevisiae Mre11-Rad50-Xrs2 Complex Stability in Addition to Nuclease Activity

The Mre11-Rad50-Xrs2 complex is involved in DNA double-strand break repair, telomere maintenance, and the intra-S phase checkpoint. The Mre11 subunit has nuclease activity in vitro, but the role of the nuclease in DNA repair and telomere maintenance remains controversial. We generated six mre11 alleles with substitutions of conserved residues within the Mre11-phosphoesterase motifs and compared the phenotypes conferred, as well as exonuclease activity and complex formation, by the mutant proteins. Substitutions of Asp16 conferred the most severe DNA repair and telomere length defects. Interactions between Mre11-D16A or Mre11-D16N and Rad50 or Xrs2 were severely compromised, whereas the mre11 alleles with greater DNA repair proficiency also exhibited stable complex formation. At all of the targeted residues, alanine substitution resulted in a more severe defect in DNA repair compared to the more conservative asparagine substitutions, but all of the mutant proteins exhibited <2% of the exonuclease activity observed for wild-type Mre11. Our results show that the structural integrity of the Mre11-Rad50-Xrs2 complex is more important than the catalytic activity of the Mre11 nuclease for the overall functions of the complex in vegetative cells.

Telomerase Inhibition and Cancer: Might Platinum Based Drugs have a Future as Anti-telomerase Pharmacological Approach?

Telomerase is a ribonucleoprotein polymerase that maintains the length of telomeric DNA by adding hexameric units (TTAGGG) to the ends of the chromosomes. This mechanism prevents replicative senescence, thus conferring unlimited proliferative potential to cells. Telomerase reactivation has been detected in most human tumour tissue, indicating that the enzyme may be useful as a specific tumour marker. The inhibition of telomerase causes a progressive and critical reduction of telomeres, leading to a potent signal for the blockage of cell proliferation and the induction of apoptosis. Since normal somatic cells lack telomerase activity, the anti-telomerase approach is highly specific for tumour cells and metastases. Prolonged treatment is required before enzyme deactivation causes the telomeres to be shortened enough to induce senescence and apoptosis. Therefore, the drugs employed in anti-telomerase therapy should be of only moderate non-specific cytotoxicity. Certain cis-Pt(II)-complexes have recently been shown to be effective inhibitors of telomerase in both cell-free and in vitro assays, most likely by targeting the nucleobases of the RNA component of the enzyme.

Chromosomal telomere shortening of kidney cells in IgA nephropathy by the measurement of DNA in urinary sediment

The histology and function of the kidney deteriorates with age and progressive renal failure, but the mechanisms involved in renal ageing are not known. In vitro studies suggest that telomere shortening is important in replicative senescence, and is accelerated by stress factors that increase replication. We investigated whether IgA nephropathy, a prototype chronic kidney disease, is associated with localized intrarenal cellular ageing. We studied the mean length of terminal restriction fragments (TRF), a measure of average telomere size, in the DNA of peripheral blood mononuclear cells and urinary sediment of 15 patients with IgA nephropathy. The mean TRF lengths in peripheral blood is 7043.8 +/- 1 182.8 base pairs, and in urinary sediment is 6 749.7 +/- 636.5 base pairs. The mean TRF lengths of urinary DNA significantly correlate with the serum creatinine (r = -0.525, p = 0.044) and estimated glomerular filtration rate (GFR) (r = 0.651, p = 0.009). The mean TRF lengths of urinary DNA had an insignificant inverse correlation with patient age (r = -0.364, p = 0.2), and do not correlate with the degree of glomerulosclerosis (r = 0.004, p = 0.9) or tubulointerstitial scarring in renal biopsy (r =-0.032, p = 0.9). After 30 months of follow-up, the rate of decline of estimated GFR has an inverse correlation with the mean TRF lengths of urinary DNA (r = -0.699, p = 0.004). The TRF lengths of peripheral blood DNA do not correlate with any clinical or histological parameter or the rate of renal function decline. Although this is a pilot study, our observation indicates that the TRF lengths of genomic DNA extracted from urinary sediment is related to the degree of renal impairment. However, a long telomere length of genomic DNA in urinary sediment is associated with a more rapid decline of renal function. Our findings might be relevant to the pathogenesis of progressive renal failure.

X‐chromosome inactivation and telomere size in newborns resulting from intracytoplasmic sperm injection

X‐chromosome inactivation and telomere size in newborns resulting from intracytoplasmic sperm injection

Erratum: Homologous recombination in Candida albicans: role of CaRad52p in DNA repair, integration of linear DNA fragments and telomere length

Erratum: Homologous recombination in <i>Candida albicans</i>: role of CaRad52p in DNA repair, integration of linear DNA fragments and telomere length

Cytoprotective effect on oxidative stress and inhibitory effect on cellular aging of Uncaria sinensis Havil

The ethanol extract from the hooks and stems of Uncaria sinensis Havil (Rubiaceae) exhibited significant inhibitory activity on oxidative stress and the age-dependent shortening of the telomeric DNA length. In the peroxidation model using t-BuOOH, the Uncaria sinensis extract showed a notable cytoprotective effect on the HEK-N/F cells with 65.0 ± 3.0% of cell viability when compared with control cells at a concentration of 50 μg/ml. In addition, the Uncaria sinensis extract exhibited a significant cytoprotective effect against UVB-induced oxidative damage. The life-span of the HEK-N/F cells was elongated by 201% as a result of the continuous administration of 3 μg/ml of the Uncaria sinensis extract compared to that of the control. These observations were attributed to the inhibitory effect of the Uncaria sinensis extract on the age-dependent shortening of the telomere length as shown by the Southern blots of the terminal restriction fragments (TRFs) of DNA extracted from subculture passages.

Cytoprotective effect on oxidative stress and inhibitory effect on cellular aging of Terminalia chebula fruit.

The ethanol extract from the fruit of Terminalia chebula (Combretaceae) exhibited significant inhibitory activity on oxidative stress and the age-dependent shortening of the telomeric DNA length. In the peroxidation model using t-BuOOH, the T. chebula extract showed a notable cytoprotective effect on the HEK-N/F cells with 60.5 +/- 3.8% at a concentration of 50 microg/ml. In addition, the T. chebula extract exhibited a significant cytoprotective effect against UVB-induced oxidative damage. The life-span of the HEK-N/F cells was elongated by 40% as a result of the continuous administration of 3 microg/ml of the T. chebula extract compared to that of the control. These observations were attributed to the inhibitory effect of the T. chebula extract on the age-dependent shortening of the telomere, length as shown by the Southern blots of the terminal restriction fragments (TRFs) of DNA extracted from subculture passages.

Genetic susceptibility to tobacco-related cancer.

Although cigarette smoking is the dominant risk factor for several epithelial cancers, only a small fraction of individuals with tobacco exposure develop cancer. The underlying hypothesis is that genetic factors may render certain smokers more susceptible to cancer than others. Genetic alterations in critical regulatory pathways may predispose cells to carcinogenesis. These pathways include regulation of xenobiotic metabolism; control of genomic stability, including DNA repair mechanisms, cell-cycle checkpoints, apoptosis and telomere length; and control of microenvironmental factors, such as matrix metalloproteinases, inflammation and growth factors. In addition, epigenetic events, such as promoter hypermethylation and loss of imprinting, are also involved in carcinogenesis. In this review, we will summarize recent advances in genetic susceptibility to tobacco-related cancer. Emphasizing on risk assessment, we will describe how genetic variations in the above-mentioned genetic pathways modify the tobacco-related cancer risk. In addition, we will discuss how genetic variations may assist in predicting clinical outcome, such as the natural history of cancer and treatment response. The measurements of genetic susceptibility by both genotypic and phenotypic assays are covered in the text. Finally, we present a number of current concerns that need to be addressed as the exciting field of molecular cancer epidemiology advances rapidly.

Homologous recombination in Candida albicans: role of CaRad52p in DNA repair, integration of linear DNA fragments and telomere length.

Chromosomal rearrangements are common in both clinical isolates and spontaneous mutants of Candida albicans. It appears that many of these rearrangements are caused by translocations around the major sequence repeat (MSR) that is present in all chromosomes except chromosome 3, suggesting that homologous recombination (HR) may play an important role in the survival of this organism. In order to gain information on these processes, we have cloned the homologue of RAD52, which in Saccharomyces cerevisiae is the only gene required for all HR events. CaRAD52 complemented poorly a rad52 mutant of S. cerevisiae. Two null Carad52Delta/Carad52Delta mutants were constructed by sequential deletion of both alleles and two reconstituted strains were obtained by reintegration of the gene. Characterization of these mutants indicated that HR plays an essential role in the repair of DNA lesions caused by both UV light and the radiomimetic compound methyl-methane-sulphonate (MMS), whereas the non-homologous end-joining pathway (NHEJ) is used only in the absence of Rad52p or after extensive DNA damage. Repair by HR is more efficient in exponentially growing than in stationary cells, probably because a larger number of cells are in late S or G2 phases of the cell cycle (and therefore, can use a sister chromatid as a substrate for recombinational repair), whereas stationary phase cells are mainly in G0 or G1, and only can be repaired using the chromosomal homologue. In addition, CaRad52p is absolutely required for the integration of linear DNA with long flanking homologous sequences. Finally, the absence of CaRad52p results in the lengthening of telomeres, even in the presence of an active telomerase, an observation not described in any other organism. This raises the possibility that both telomerase and homologous recombination may function simultaneously at C. albicans telomeres.

Assessment of Telomere Length, Phenotype, and DNA Content

AbstractTelomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently‐labeled probes (Flow‐FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen‐antibody complexes are covalently cross‐linked onto cell membranes before FISH with a telomere‐specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow‐FISH. Telomere fluorescence of G0/1 cells of subpopulations and internal standards obtained from Flow‐FISH are normalized for DNA ploidy and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length.

Engineering anticancer T cells for extended functional longevity.

Like other somatic cells, human T lymphocytes have a finite replicative capacity in vitro, and, by implication and consistent with the limited data available, in vivo as well. An accumulation of dysfunctional T cells may be detrimental under conditions of chronic antigenic stress (chronic infection, cancer, autoimmunity). Using T cells from young donors to model the process of T cell clonal expansion in vitro under these conditions reveals age-associated increasing levels of oxidative DNA damage and microsatellite instability (MSI), coupled with decreasing DNA repair capacity, telomerase induction and telomere length, decreased levels of expression of the T cell costimulator CD28 and consequently reduced secretion of the T cell growth factor interleukin-2 (IL-2). However, data from similar experiments using T cell clones (TCCs) derived from extremely healthy very elderly donors ("successfully aged") indicate that DNA repair is better maintained, MSI less prevalent, and (already short) telomere lengths are maintained. Nonetheless, oxidative DNA damage is seen to the same extent, and clonal longevity is also similar in these clones. DNA damage levels are reduced by culture in 5% oxygen, but longevity is not improved. This may be because of the requirement for intermittent reactivation via receptor pathways dependent on free radical production in T cells. These recent findings from our international immunosenescence research consortium suggest that strategies other than telomere maintenance, better protection against free radicals, or improved DNA repair will be required for functional longevity extension of human TCCs. To obtain sufficient cells for adoptive immunotherapy of cancer, alternative avenues need exploration; currently, these include enforced expression of certain heat shock proteins and proteasome components, and interference with the expression of negative regulatory receptors expressed by T cells.

Shorter telomeres, accelerated ageing and increased lymphoma in DNA‐PKcs‐deficient mice

Non-homologous end joining (NHEJ) is the principal repair mechanism used by mammalian cells to cope with double-strand breaks (DSBs) that continually occur in the genome. One of the key components of the mammalian NHEJ machinery is the DNA-PK complex, formed by the Ku86/70 heterodimer and the DNA-PK catalytic subunit (DNA-PKcs). Here, we report on the detailed life-long follow-up of DNA-PKcs-defective mice. Apart from defining a role of DNA-PKcs in telomere length maintenance in the context of the ageing organism, we observed that DNA-PKcs-defective mice had a shorter life span and showed an earlier onset of ageing-related pathologies than the corresponding wild-type littermates. In addition, DNA-PKcs ablation was associated with a markedly higher incidence of T lymphomas and infections. In conclusion, these data link the dual role of DNA-PKcs in DNA repair and telomere length maintenance to organismal ageing and cancer.

Telomeres and cardiovascular disease: does size matter?

Telomeres-the specialized DNA-protein structures at the ends of eukaryotic chromosomes-are essential for maintaining genome stability and integrity and for extended proliferative life span in both cultured cells and in the whole organism. Telomerase and additional telomere-associated proteins are necessary for preserving telomeric DNA length. Age-dependent telomere shortening in most somatic cells, including vascular endothelial cells, smooth muscle cells, and cardiomyocytes, is thought to impair cellular function and viability of the aged organism. Telomere dysfunction is emerging as an important factor in the pathogenesis of hypertension, atherosclerosis, and heart failure. In this Review, we discuss present studies on telomeres and telomere-associated proteins in cardiovascular pathobiology and their implications for therapeutics.

Rapid telomere attrition in cardiac tissue of the ageing Wistar rat

Many studies show an association between ageing and mean telomere length in DNA isolated from peripheral blood mononuclear cells, few studies have examined less accessible tissues. This study has two objectives: (i) to define the best method to prepare rodent DNA for telomere length measurement by Southern blotting and (ii) to determine whether there are differential rates of telomere attrition in different rodent tissues. We found that the use of agarose plugs for DNA isolation was essential for the accurate measurement of rodent telomere length. Tissue was collected from neonatal (3 days) or aged (18–24 months) male Wistar rats and terminal restriction fragment (TRF) length was measured by Southern blotting. Cardiac tissue from aged rats showed a 38% loss of TRF length compared with newborn animals ( p<0.001, n=13), this contrasts with much smaller reductions in brain (1.6%), liver (14.2%), kidney (8.9%) and lung (9.7%). This study demonstrates that the methods of DNA preparation are critical for accurate measurement of telomeres in rodent tissues. Moreover, we show differential rates of telomere attrition in rat tissues, the heart being most susceptible to telomere loss. These observations could have important implications for the study of age-specific changes in tissue function.

Importance of TRF1 for Functional Telomere Structure

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.

A novel allele of fission yeast rad11 that causes defects in DNA repair and telomere length regulation.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11(+) gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and gamma-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.

DNA Binding and Telomere Length Regulation of Yeast RAP1 Homologues

The repressor activator protein 1 ( RAP1) has many important functions in Saccharomyces cerevisiae. At the chromosome ends, it is a negative regulator of telomere length. Here, we show that Saccharomyces castellii/ Saccharomyces dairensis telomeric sequences inserted into a S. cerevisiae telomere are counted as part of the telomere, consistent with the presence of high-affinity Rap1p binding sites within these sequences. We show that S. castellii Rap1p (scasRap1p) can regulate telomere length in a S. cerevisiae strain, albeit less stringently. Cloning of the S. dairensis RAP1 homologue ( sdaiRAP1) revealed that it encodes the largest RAP1 protein identified to date. Despite its large size, binding analyses of the recombinant sdaiRap1p revealed that the protein binds with the same spacing and with similar affinity to yeast telomeric sequences, as the scer- and scasRAP1 proteins. According to the Rap1p counting model for telomere length regulation, a low density of Rap1p binding sites in a telomere would result in a longer telomere in S. cerevisiae . We have compared the lengths of two individual S. dairensis telomeres and find that their lengths are not regulated to give the same number of high-affinity binding sites. This may be due to other factors than Rap1p having influence on the telomere length regulation.

Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae

In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length [Curr. Opin. Cell Biol. 13 (2001) 281]. Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex). Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage [Genes Dev. 15 (2001) 2238; Mol. Cell 7 (2001) 1255]. We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p. Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents. We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases. In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p. We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres. As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.

Shortening of telomeres: Evidence for replicative senescence of T cells derived from patients with Wegener's granulomatosis

Replicative senescence describes the fact that somatic cells undergo a finite and predictable number of cell divisions before entering an irreversible state of growth arrest. Progressive shortening of the telomeres, a consequence of cell division, is a reliable indicator of replicative senescence. We analyzed telomere length of DNA derived from T cells of patients suffering from Wegener's granulomatosis by Southern blotting. Moreover, expression of CD28, another marker for replicative senescence, was tested by cytofluorometry. In patients with disease for more than 5 years, short telomeres were detected in addition to telomeres of normal length, indicating replicative senescence of discrete T-cell clones. Reduced expression of CD28 was noted, particularly on CD8-positive T cells, derived from patients with disease for more than 5 years and short telomeres. Our data provide evidence that a portion of T cells had undergone replicative senescence, which in turn indicates clonal expansion of T cells as consequence of activation.