Stabilization of telomere length and karyotypic stability are directly correlated with the level of hTERT gene expression in primary fibroblasts

Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase (hTERT) gene in sheep fibroblasts reconstitutes telomerase activity and extends their lifespan. However, telomere length is not maintained in all cell lines, even though in vitro telomerase activity is restored in all of them. Cell lines expressing higher levels of hTERT mRNA do not exhibit telomere erosion or genomic instability. By contrast, fibroblasts expressing lower levels of hTERT do exhibit telomere shortening, although the telomeres eventually stabilize at a shorter length. The shorter telomere lengths and the extent of karyotypic abnormalities are both functions of hTERT expression level. We conclude that telomerase activity is required to bypass senescence but is not sufficient to prevent telomere erosion and genomic instability at lower levels of expression.     

Methods of studying telomere damage induced by quadruplex-ligand complexes

The burgeoning knowledge about the structure of telomeres and the roles of various factors involved in telomere maintenance provides several possible targets for pharmacological intervention. To date the area that has received major attention regarding drug discovery is the targeting the telomeric G-quadruplex (G4) structure. G4 ligands were initially designed to counteract telomerase action at telomeres. Surprisingly, their antiproliferative effects can occur in telomerase negative cells and follow kinetics, which cannot be merely explained by telomere shortening, suggesting that these compounds affect other pathways, not necessarily related to telomere biology. Impressively, it has been shown that polyaromatic compounds featuring end-stacking binding properties trigger a strong DNA damage response at telomeres. This is typical of the telomere deprotection occurring during cellular senescence or upon telomere injury. It emerged that the G4-interacting agents are more than simple telomerase inhibitors and that their direct target is rather telomere than telomerase. This review summarizes the most valid experimental approaches for studying the pharmacological telomere damage induced by G4-ligand complexes.     

Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span

The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.     

Telomerase maintains telomere structure in normal human cells

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.     

Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro

Although vertebrate telomeres are highly conserved, telomere dynamics and telomerase profiles vary among species. The objective of the present study was to examine telomerase activity and telomere length profiles of transformed and non-transformed avian cells in vitro. Non-transformed chicken embryo fibroblasts (CEFs) showed little or no telomerase activity from the earliest passages through senescence. Unexpectedly, a single culture of particularly long-lived senescent CEFs showed telomerase activity after over 250 days in culture. Transformed avian lines (six chicken, two quail and one turkey) and tumor samples (two chicken) exhibited telomerase activity. Telomere length profiles of non-transformed CEF cultures derived from individual embryos of an inbred line (UCD 003) exhibited cycles of shortening and lengthening with a substantial net loss of telomeric DNA by senescence. The telomere length profiles of several transformed cell lines resembled telomere length profiles of senescent CEFs in that they exhibited little of the typical smear of terminal restriction fragments (TRFs) suggesting that these transformed cells may possess a reduced amount of telomeric DNA. These results show that avian telomerase activity profiles are consistent with the telomerase activity profiles of human primary and transformed cells. Further, monitoring of telomere lengths of primary cells provides evidence for a dynamic series of changes over the lifespan of any specific cell culture ultimately resulting in net telomeric DNA loss by senescence.     

Role of telomere in endothelial dysfunction in atherosclerosis

PURPOSE OF REVIEW: Telomeres consist of repeats of G-rich sequence at the end of chromosomes. These DNA repeats are synthesized by enzymatic activity associated with an RNA protein complex called telomerase. In most somatic cells, telomerase activity is insufficient, and telomere length decreases with increasing cell division, resulting in an irreversible cell growth arrest, termed cellular senescence. Cellular senescence is associated with an array of phenotypic changes suggestive of aging. Until recently, cellular senescence has largely been studied as an in-vitro phenomenon; however, there is accumulating evidence that indicates a critical role of telomere function in the pathogenesis of human atherosclerosis. This review attempts to summarize recent work in vascular biology that supports the "telomere hypothesis". We discuss the possible relevance of telomere function to vascular aging and the therapeutic potential of telomere manipulation. RECENT FINDINGS: It has been reported that many of the changes in senescent vascular cell behavior are consistent with known changes seen in age-related vascular diseases. Introduction of telomere malfunction has been shown to lead to endothelial dysfunction that promotes atherogenesis, whereas telomere lengthening extends cell lifespan and protects against endothelial dysfunction associated with senescence. Indeed, recent studies have demonstrated that telomere attrition and cellular senescence occur in the blood vessels and are associated with human atherosclerosis. SUMMARY: Recent findings suggest that vascular cell senescence induced by telomere shortening may contribute to atherogenesis and may provide insights into a novel treatment of antisenescence to prevent atherosclerosis.     

A role for sister telomere cohesion in telomere elongation by telomerase

Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC) a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit TIN2. The DC-TIN2 mutations were clustered in small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and further, that HP1 binding to TIN2 was required for sister telomere cohesion in S phase and for telomere length maintenance by telomerase. We briefly review and discuss the implications of our findings in this Extra View, and present some new data that may shed light on how sister telomere cohesion could influence telomere elongation by telomerase.     

Clonal heterogeneity in telomerase activity and telomere length in tumor-derived cell lines

The ribonucleoprotein, telomerase, is responsible for the maintenance of telomere length in most immortal and cancer cells. Telomerase appears to be a marker of human malignancy with at least 85% of human cancers expressing its activity. In the present study, we examined a series of tumor-derived and in vitro immortalized cell lines for telomerase activity levels, telomere lengths, and expression levels of the RNA and catalytic components of telomerase. We found significant variability in both telomere lengths and telomerase activity in clones from tumor cells. In addition, the levels of telomerase components or telomerase activity were not predictive of telomere length. Data from clonally derived cells suggest that critically shortened telomeres in these tumor-derived cell lines may signal activation of telomerase activity through an increase in the expression of the catalytic subunit of telomerase. Although clones with low telomerase shorten their telomeres over time, their subclones all have high levels of telomerase activity with no telomere shortening. In addition, analysis of early clones for telomerase activity indicates substantial variability, which suggests that activity levels fluctuate in individual cells. Our data imply that cell populations exhibit a cyclic expression of telomerase activity, which may be partially regulated by telomere shortening.     

A role for sister telomere cohesion in telomere elongation by telomerase

Telomere length homeostasis is achieved by a balance of telomere shortening caused by DNA replication and nucleolytic attack and telomere lengthening by telomerase. The importance of telomere length maintenance to human health is best illustrated by dyskeratosis congenita (DC), a disease of telomere shortening caused by mutations in telomerase subunits. DC patients suffer stem cell depletion and die of bone marrow stem cell failure. Recently a new class of particularly severe DC patients was found to harbor mutations in the shelterin subunit TIN2. The DC-TIN2 mutations were clustered in small domain of unknown function. In a recently published study we showed that the DC mutation cluster in TIN2 harbored a binding site for heterochromatin protein 1 (HP1) and, further, that HP1 binding to TIN2 was required for sister telomere cohesion in S phase and for telomere length maintenance by telomerase. We briefly review and discuss the implications of our findings in this Extra View and present some new data that may shed light on how sister telomere cohesion could influence telomere elongation by telomerase.Key words: telomeres, cohesion, telomerase, TIN2, dyskeratosis congenita     

Association of telomere instability with senescence of porcine cells

Telomeres are essential for the maintenance of genomic stability, and telomere dysfunction leads to cellular senescence, carcinogenesis, aging, and age-related diseases in humans. Pigs have become increasingly important large animal models for preclinical tests and study of human diseases, and also may provide xeno-transplantation sources. Thus far, Southern blot analysis has been used to estimate average telomere lengths in pigs. Telomere quantitative fluorescence in situ hybridization (Q-FISH), however, can reveal status of individual telomeres in fewer cells, in addition to quantifying relative telomere lengths, and has been commonly used for study of telomere function of mouse and human cells. We attempted to investigate telomere characteristics of porcine cells using telomere Q-FISH method. The average telomere lengths in porcine cells measured by Q-FISH correlated with those of quantitative real-time PCR method (qPCR) or telomere restriction fragments (TRFs) by Southern blot analysis. Unexpectedly, we found that porcine cells exhibited high incidence of telomere doublets revealed by Q-FISH method, coincided with increased frequency of cellular senescence. Also, telomeres shortened during subculture of various porcine primary cell types. Interestingly, the high frequency of porcine telomere doublets and telomere loss was associated with telomere dysfunction-induced foci (TIFs). The incidence of TIFs, telomere doublets and telomere loss increased with telomere shortening and cellular senescence during subculture. Q-FISH method using telomere PNA probe is particularly useful for characterization of porcine telomeres. Porcine cells exhibit high frequency of telomere instability and are susceptible to telomere damage and replicative senescence.