Telomere Restoration Research Articles

Thymidine nucleotide metabolism controls human telomere length.

Telomere length in humans is associated with lifespan and severe diseases, yet the genetic determinants of telomere length remain incompletely defined. Here we performed genome-wide CRISPR-Cas9 functional telomere length screening and identified thymidine (dT) nucleotide metabolism as a limiting factor in human telomere maintenance. Targeted genetic disruption using CRISPR-Cas9 revealed multiple telomere length control points across the thymidine nucleotide metabolism pathway: decreasing dT nucleotide salvage via deletion of the gene encoding nuclear thymidine kinase (TK1) or de novo production by knockout of the thymidylate synthase gene (TYMS) decreased telomere length, whereas inactivation of the deoxynucleoside triphosphohydrolase-encoding gene SAMHD1 lengthened telomeres. Remarkably, supplementation with dT alone drove robust telomere elongation by telomerase in cells, and thymidine triphosphate stimulated telomerase activity in a substrate-independent manner in vitro. In induced pluripotent stem cells derived from patients with genetic telomere biology disorders, dT supplementation or inhibition of SAMHD1 promoted telomere restoration. Our results demonstrate a critical role of thymidine metabolism in controlling human telomerase and telomere length, which may be therapeutically actionable in patients with fatal degenerative diseases.

New Genetic Bomb Trigger: Design, Synthesis, Molecular Dynamics Simulation, and Biological Evaluation of Novel BIBR1532-Related Analogs Targeting Telomerase against Non-Small Cell Lung Cancer.

Telomeres serve a critical function in cell replication and proliferation at every stage of the cell cycle. Telomerase is a ribonucleoprotein, responsible for maintaining the telomere length and chromosomal integrity of frequently dividing cells. Although it is silenced in most human somatic cells, telomere restoration occurs in cancer cells because of telomerase activation or alternative telomere lengthening. The telomerase enzyme is a universal anticancer target that is expressed in 85–95% of cancers. BIBR1532 is a selective non-nucleoside potent telomerase inhibitor that acts by direct noncompetitive inhibition. Relying on its structural features, three different series were designed, and 30 novel compounds were synthesized and biologically evaluated as telomerase inhibitors using a telomeric repeat amplification protocol (TRAP) assay. Target compounds 29a, 36b, and 39b reported the greatest inhibitory effect on telomerase enzyme with IC50 values of 1.7, 0.3, and 2.0 μM, respectively, while BIBR1532 displayed IC50 = 0.2 μM. Compounds 29a, 36b, and 39b were subsequently tested using a living-cell TRAP assay and were able to penetrate the cell membrane and inhibit telomerase inside living cancer cells. Compound 36b was tested for cytotoxicity against 60 cancer cell lines using the NCI (USA) procedure, and the % growth was minimally impacted, indicating telomerase enzyme selectivity. To investigate the interaction of compound 36b with the telomerase allosteric binding site, molecular docking and molecular dynamics simulations were used.

The tarnished silver spoon? Trade-off between prenatal growth and telomere length in wild boar.

Life‐history theory predicts a trade‐off between growth rates and lifespan, which is reflected by telomere length, a biomarker of somatic state. We investigated the correlation between telomere length and early‐life growth of wild boar piglets, Sus scrofa, kept under semi‐natural conditions with high food availability to examine our hypothesis that increased pre‐ and postnatal growth will lead to telomere length attrition, but that a high supply of nutrient may provide the possibility to compensate telomere loss via telomere repair mechanisms. As predicted, our data showed a clear negative correlation between birth body mass and initial telomere length: heavier neonates had shorter telomeres at birth, and we did not find an influence of the mother on initial telomere length. Body mass at birth correlated with body mass later in life and postnatal growth rate did not affect telomere length. We observed an increase in telomere length during postnatal development, suggesting that high food availability allowed piglets to invest into both, growth and telomere restoration. The increase in telomere length over the duration of the study was not accompanied by telomerase activity; thus, telomere elongation was caused either by alternative mechanisms or by short pulses of telomerase activity that we missed. Taken together, this study suggests a trade‐off between investment into growth and telomere maintenance even before birth and the possibility to compensate telomere attrition during growth under high amounts of available energy.

Small-Molecule PAPD5 Inhibitors Restore Telomerase Activity in Patient Stem Cells.

Genetic lesions that reduce telomerase activity inhibit stem cell replication and cause a range of incurable diseases, including dyskeratosis congenita (DC) and pulmonary fibrosis (PF). Modalities to restore telomerase in stem cells throughout the body remain unclear. Here, we describe small-molecule PAPD5 inhibitors that demonstrate telomere restoration invitro, in stem cell models, and invivo. PAPD5 is a non-canonical polymerase that oligoadenylates and destabilizes telomerase RNA component (TERC). We identified BCH001, a specific PAPD5 inhibitor that restored telomerase activity and telomere length in DC patient induced pluripotent stem cells. When human blood stem cells engineered to carry DC-causing PARN mutations were xenotransplanted into immunodeficient mice, oral treatment with a repurposed PAPD5 inhibitor, the dihydroquinolizinone RG7834, rescued TERC 3' end maturation and telomere length. These findings pave the way for developing systemic telomere therapeutics to counteract stem cell exhaustion in DC, PF, and possibly other aging-related diseases.

Oncological role of HMGA2 (Review)

The high mobility groupA2 (HMGA2) protein is a non‑histone architectural transcription factor that modulates the transcription of several genes by binding to AT‑rich sequences in the minor groove of B‑form DNA and alters the chromatin structure. As a result, HMGA2 influences a variety of biological processes, including the cell cycle process, DNA damage repair process, apoptosis, senescence, epithelial‑mesenchymal transition and telomere restoration. In addition, the overexpression of HMGA2 is a feature of malignancy, and its elevated expression in human cancer predicts the efficacy of certain chemotherapeutic agents. Accumulating evidence has suggested that the detection of HMGA2 can be used as a routine procedure in clinical tumour analysis.

Perfluorinated substances and telomeres in an Arctic seabird: Cross-sectional and longitudinal approaches

Telomeres are non-coding DNA repeats located at the termini of eukaryotic chromosomes, regulated by dynamic processes balancing shortening and maintenance. Despite a mechanism to slow-down telomere shortening, cell division leads to progressive attrition of chromosomes, leading to the onset of cellular senescence or apoptosis. However, telomere restoration based on telomerase activity is the primary mechanism for telomere maintenance. Telomere length is associated to health and survival and can be impacted by a broad panel of environmental factors. However, the effect of contaminants on telomeres is poorly known for living organisms. The aim of this study was to investigate relationships between some poly- and perfluoroalkyl substances (PFASs), body condition and telomere length by using both a cross-sectional and longitudinal approach in adult breeding Black-legged kittiwakes (Rissa tridactyla) from Svalbard. First, we examined the associations between absolute telomere length and PFASs contamination in a given year (cross-sectional approach). Second, we investigated the relationships between telomere dynamics and PFASs contamination within a two years’ time frame (longitudinal approach). Our results did not show any significant relationships of PFASs and body condition with absolute telomere length in a given year. Surprisingly, we found a positive and significant relationship between PFASs and telomere dynamics in both sexes with elongated telomere in birds bearing the highest concentrations of PFASs. Our study underlines (i) the need to investigate PFAS effects on telomere dynamics with a longitudinal approach and (ii) a potential positive effect of these contaminants on telomere length, with the most contaminated birds showing the slowest rate of telomere shortening or even displaying elongated ones. Our study is the first to report a relationship between PFASs and telomere length in free-living vertebrates. A possible underlying mechanism and other potential confounding factors are discussed.

Mathematical analysis of a clonal evolution model of tumour cell proliferation

We investigate a partial differential equation model of a cancer cell population, which is structured with respect to age and telomere length of cells. We assume a continuous telomere length structure, which is applicable to the clonal evolution model of cancer cell growth. This model has a non-standard non-local boundary condition. We establish global existence of solutions and study their qualitative behaviour. We study the effect of telomere restoration on cancer cell dynamics. Our results indicate that without telomere restoration, the cell population extinguishes. With telomere restoration, exponential growth occurs in the linear model. We further characterise the specific growth behaviour of the cell population for special cases. We also study the effects of crowding induced mortality on the qualitative behaviour, and the existence and stability of steady states of a nonlinear model incorporating crowding effect. We present examples and extensive numerical simulations, which illustrate the rich dynamic behaviour of the linear and nonlinear models.

Changes of telomere status with aging: An update.

Accumulated data have shown that most human somatic cells or tissues show irreversible telomere shortening with age, and that there are strong associations between telomere attrition and aging-related diseases, including cancers, diabetes and cognitive disorders. Although it has been largely accepted that telomere attrition is one of the major causes of aging-related disorders, critical aspects of telomere biology remain unresolved, especially the lack of standardized methodology for quantification of telomere length. Another frustrating issue is that no potentially promising methods for safe prevention of telomere erosion, or for telomere elongation, have been devised. Here, we review several methods for quantification of telomere length currently utilized worldwide, considering their advantages and drawbacks. We also summarize the results of our recent studies of human cells and tissues, mainly using quantitative fluorescence in situ hybridization and Southern blotting, including those derived from patients with progeria-prone Werner syndrome and trisomy 21, and several strains of induced pluripotent stem cells. We discuss the possible merits of using telomere shortness as an indicator, or a new marker, for diagnosis of precancerous states and aging-related disorders. In addition, we describe newly found factors that are thought to impact telomere dynamics, providing a new avenue for examining the unsolved issues related to telomere restoration and maintenance.

Stress exposure in early post-natal life reduces telomere length: an experimental demonstration in a long-lived seabird

Exposure to stressors early in life is associated with faster ageing and reduced longevity. One important mechanism that could underlie these late life effects is increased telomere loss. Telomere length in early post-natal life is an important predictor of subsequent lifespan, but the factors underpinning its variability are poorly understood. Recent human studies have linked stress exposure to increased telomere loss. These studies have of necessity been non-experimental and are consequently subjected to several confounding factors; also, being based on leucocyte populations, where cell composition is variable and some telomere restoration can occur, the extent to which these effects extend beyond the immune system has been questioned. In this study, we experimentally manipulated stress exposure early in post-natal life in nestling European shags (Phalacrocorax aristotelis) in the wild and examined the effect on telomere length in erythrocytes. Our results show that greater stress exposure during early post-natal life increases telomere loss at this life-history stage, and that such an effect is not confined to immune cells. The delayed effects of increased telomere attrition in early life could therefore give rise to a ‘time bomb’ that reduces longevity in the absence of any obvious phenotypic consequences early in life.

36 RESTORATION OF TELOMERE LENGTH IN CLONED PIG EMBRYOS DURING EARLY EMBRYOGENESIS IS NOT DEPENDENT ON TELOMERE LENGTH AND TYPE OF DONOR CELLS

Analyses on telomere length in cloned animals have revealed diverse results depending on the donor cell types. In mice and cattle, telomere length is reset during morula-blastocyst transition and the restoration is thought to be a telomerase-dependent process. However, it is still unknown whether the pattern of telomere elongation during this transition is dependent on donor cell types. In the present study, we examined the changes of telomere length during morula-blastocyst transition in cloned porcine embryos using different types of donor cell. Embryonic stem-like cells (ES), cumulus cells (C), fibroblasts at passages 7 and 10 (F7 and F10, respectively) were used as donor cells to produce NT embryos (ES, C, F7, and F10 groups, respectively). Telomere lengths of ES (35.8 ± 1.5 kb), C (24.4 ± 0.5 kb), P7 (18.7 ± 0.6 kb), and P10 (17.2 ± 0.1 kb) cells were significantly different. In contrast, cloned morulae in ES, C, F7, and F10 groups did not have any significant differences in telomere length (18.2 ± 0.3, 17.8 ± 0.7, 18.5 ± 0.3, and 18.4 ± 0.4 kb, respectively). Likewise, cloned blastocysts in ES, C, F7, and F10 groups had similar telomere length (22.3 ± 1.5, 23.5 ± 2.6, 20.2 ± 1.0, and 20.9 ± 1.0 kb, respectively). However, the telomere of the blastocyst was significantly longer (P < 0.05) compared with the morula in the respective group. Furthermore, relative telomerase activities of cloned morulae in ES, C, F7, and F10 groups (4.2 ± 0.4, 4.0 ± 0.5, 5.1 ± 0.4, and 4.9 ± 0.4, respectively) were significantly lower (P < 0.01) than those of cloned blastocysts in the same groups (8.2 ± 1.1, 8.6 ± 0.6, 12.5 ± 2.9, and 8.3 ± 1.1, respectively). The proportions of blastocysts in cloned embryos for ES, C, F7, and F10 groups (10.0 ± 5.2, 17.3 ± 2.9, 13.5 ± 2.9, and 13.1 ± 3.6%, respectively) did not significantly differ. Total cell numbers in blastocysts for ES, C, F7, and F10 groups (28.3 ± 2.9, 32.6 ± 3.6, 30.4 ± 3.1, and 27.4 ± 2.2, respectively) were not significantly different as well. In the present study, we found that the telomere elongation in cloned pig embryos occurs during morula-blastocyst transition. This is consistent with the previous findings in in vivo and in vitro fertilization and cloned embryos in cattle and mice. We also revealed that although different types of cells (ES, C, and F) or the same type of cells with different telomere length (F7 and F10) were used for NT, their resultant morulae and blastocysts had similar telomere length. This suggests that the telomere restoration during morula-blastocyst transition is independent of telomere length and type of donor cells. An increase in telomerase activity during morula-blastocyst transition indicates that the elongation of telomere length was likely a telomerase-dependent process. In conclusion, restoration of telomere length in cloned porcine embryos during morula-blastocyst transition was independent of telomere length and type of donor cells, and likely a telomerase-dependent process.

Molecular Biology: Telomerase Tells on Lifestyle

Being overweight, stressed, and sedentary have all been shown to accelerate the shortening of telomeres, DNA–protein complexes at the end of chromosomes that protect the genetic material and promote chromosomal stability. A new pilot study now finds that certain lifestyle changes may promote telomere integrity and length by significantly boosting telomerase activity in human immune cells. Chromosomal stability is strongly correlated with longevity. Thus, if telomere length decreases prematurely, so also does life span. Shortened telomeres have been associated with a heightened disease risk (e.g., a 100-fold higher incidence of vascular dementia) and increased mortality rates for cancer and heart disease. Moreover, evidence published in the March–April 2006 issue of Urologic Oncology indicates that by initiating chromosomal instability, short dysfunctional telomeres may participate in prostate carcinogenesis. The leading hypothesis is that telomere attrition is influenced by oxidative stress (which can result from excessive body weight and stress as well as exposure to radiation and certain pollutants, such as airborne particles) and by inflammation (which generates and is perpetuated by oxidative stress). Telomerase, an enzyme that directs the replication of telomeres, adds telomeric repeat sequences to the chromosomal DNA ends, explains principal investigator Elizabeth Blackburn, a molecular biologist at the University of California, San Francisco. “This preserves not only telomere length, but in the case of cells of the immune system, also healthy cell function and long-term immune function.” Nevertheless, she adds, “We do not yet know whether increasing telomerase activity could help reverse the problem of age-related telomere shortening.” The study, reported online 15 September 2008 ahead of print in Lancet Oncology, assessed whether lifestyle changes could increase telomerase activity in peripheral blood mononuclear cells (PBMCs), an infection-fighting component of the immune system. The authors hypothesized that improvements in nutrition, exercise, and stress management—lifestyle factors thought to help prevent cancer and cardiovascular disease—might also bolster telomerase function. Thirty men with biopsy-diagnosed, low-risk prostate cancer attended a three-day retreat followed by an outpatient phase. The men were asked to make comprehensive lifestyle changes: 1) adhere to a low-fat, primarily vegetarian diet supplemented with soy protein, fish oil, selenium, and vitamins C and E; 2) engage in moderate aerobic activity 30 minutes per day, 6 days a week; 3) practice stress management techniques such as yoga stretching, breathing, and meditation 6 days a week; and 4) attend a 1-hour support group once a week. Adherence was assessed by questionnaire. Changes in telomerase activity were measured at baseline and after 3 months. Among 24 patients with sufficient PBMCs for longitudinal analysis, two-thirds showed a significant (29–84%) increase in telomerase activity, an indication of telomere restoration. This increase was also associated with significant decreases in low-density lipoprotein cholesterol and in one measure of psychological distress (intrusive thoughts). There were no significant changes in total levels of prostate-specific antigen, the marker for prostate cancer, nor was there evidence of cancer progression in the group. “It seems remarkable that decreased stress and improved diet might give rise to measurable increases in telomerase activity in such a short time period,” says University of Glasgow biologist Pat Monaghan, who with Mark Haussmann coauthored a report on lifestyle and telomere dynamics in the January 2006 issue of Trends in Ecology & Evolution. Robert Sapolsky, a neuroendocrinologist at Stanford University, adds that the study “shows that [an] undesirable telomerase profile can be reversed with the sorts of lifestyle interventions that decrease the risk of various stress-related diseases.” The potential confounding factors and limitations of the study—the small size and short duration of the study, the complexity of the lifestyle changes, the absence of any data on the effect on telomere length, and the lack of an independent control group—make it impossible to identify causal factors. “Since these lifestyle changes and biological factors all tend to interact and reinforce each other, it’s difficult to determine which matter most,” says coauthor Elissa Epel, an associate professor of psychiatry at the University of California, San Francisco. Moreover, says Monaghan, the study involved a broad age range of men, and it is unknown whether the effects varied with the age of the patient. Nevertheless, says Epel, the findings point to some provocative possibilities. “If we can improve our health behaviors, we might be able to reverse telomere shortening or at least stabilize the telomeres in our blood,” she says. Parsing out the most critical parts of the intervention is an important direction for future research. For now, Epel says, the take-home message is that what’s good for promoting cardiovascular health also seems to help curb cellular aging. This knowledge, in turn, could help motivate people to change their diet, exercise, and stress management habits for the better.

Telomere restoration and extension of proliferative lifespan in dyskeratosis congenita fibroblasts

Dyskeratosis congenita (DC), an inherited bone marrow failure syndrome, is caused by defects in telomerase. Somatic cells from DC patients have shortened telomeres and clinical symptoms are most pronounced in organs with a high cell turnover, including those involved in hematopoiesis and skin function. We previously identified an autosomal dominant (AD) form of DC that is caused by mutations in the telomerase RNA component (TER). In this study, we evaluated whether retroviral expression of TER and/or telomerase reverse transcriptase (TERT), the catalytic component of telomerase, could extend telomere length and rescue AD DC cells from a phenotype characteristic of early senescence. Exogenous TER expression, without TERT, could not activate telomerase in AD DC skin fibroblasts. Transduction of TERT alone, however, provided AD DC cells with sufficient telomerase activity to extend average telomere length and proliferative capacity. Interestingly, we found that expression of TER and TERT together resulted in extension of lifespan and higher levels of telomerase and longer telomeres than expression of TERT alone in both AD DC and normal cells. Our results provide evidence that AD DC cells can be rescued from defects in telomere maintenance and proliferation, and that coexpression of TERT and TER together provides a more efficient means to elongate telomeres than expression of TERT alone. Similar strategies may be useful for ameliorating the detrimental effects of telomere shortening in AD DC and other diseases associated with telomerase or telomere defects.

Telomerase maintained in self-renewing tissues during serial regeneration of the urochordate Botryllus schlosseri

Telomerase is critical for the protection of germ line and stem cell chromosomes from fatal shortening during replication. In most organisms, telomerase activity is suppressed in progressively committed cells and falls to basal rates in terminally differentiated lineages. The colonial ascidian Botryllus schlosseri propagates asexually and sexually, presumably from pools of stem cells that self-renew throughout the 2- to 5-year colony life span. Asexual budding takes place continuously from the parental body wall. When the colony reaches a critical size, sexual reproduction commences with the generation of gonads. Here, we establish the existence of 6–15 kb telomeres on the ends of Botryllus chromosomes. We develop a real-time quantitative PCR telomeric repeat amplification protocol (TRAP) assay that reliably detects 0.2–100 TPG units in cells and tissues. We find highest levels of enzymatic activity in the gonads, developing embryos, and tissues containing the earliest asexual buds. Telomerase activity appears to be suppressed in later buds during organogenesis and falls to basal rates in mature zooids. We postulate that this pattern reflects maximum telomere restoration in somatic stem cells of early buds and suppression of telomerase activity in progenitors and terminally differentiated cells, indicative of an alternate role for stem cells as repeated body regenerators in colonial life histories.