scholarly journals Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism

Science ◽  
2017 ◽  
Vol 357 (6358) ◽  
pp. 1416-1420 ◽  
Author(s):  
Kunitoshi Chiba ◽  
Franziska K. Lorbeer ◽  
A. Hunter Shain ◽  
David T. McSwiggen ◽  
Eva Schruf ◽  
...  
Keyword(s):  
Genome Instability ◽  
Telomere Maintenance ◽  
Second Phase ◽  
Telomere Shortening ◽  
Telomere Attrition ◽  
Cellular Life ◽  
Two Phases ◽  
Promoter Mutations ◽  
Step Mechanism

TERT promoter mutations (TPMs) are the most common noncoding mutations in cancer. The timing and consequences of TPMs have not been fully established. Here, we show that TPMs acquired at the transition from benign nevus to malignant melanoma do not support telomere maintenance. In vitro experiments revealed that TPMs do not prevent telomere attrition, resulting in cells with critically short and unprotected telomeres. Immortalization by TPMs requires a gradual up-regulation of telomerase, coinciding with telomere fusions. These data suggest that TPMs contribute to tumorigenesis by promoting immortalization and genomic instability in two phases. In an initial phase, TPMs do not prevent bulk telomere shortening but extend cellular life span by healing the shortest telomeres. In the second phase, the critically short telomeres lead to genome instability and telomerase is further up-regulated to sustain cell proliferation.

Implication of Telomerase in Functions Besides Telomere Elongation in Human Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-40-SCI-40
Author(s):  
Francesca Gazzaniga ◽  
Jue Lin ◽  
Elizabeth Blackburn
Keyword(s):  
Cell Proliferation ◽  
Cancer Cells ◽  
Core Protein ◽  
Human Cells ◽  
Telomere Maintenance ◽  
Telomere Shortening ◽  
Telomere Attrition ◽  
Independent Functions ◽  
Equity Ownership

Abstract Abstract SCI-40 The ribonucleoprotein enzyme telomerase, which counteracts telomere shortening by adding telomeric DNA repeats to the 3’ ends of chromosomes, contains a core protein reverse transcriptase (called hTERT in humans), the essential template-containing RNA (hTER) and associated factors. Thus, telomerase is capable of circumventing the limit on cell division imposed by telomere attrition. Telomerase enzymatic activity is readily detectable in stem and progenitor cell populations, but is largely downregulated in most adult somatic human cells. In contrast, telomerase is highly active in the large majority (∼80%–90%) of cancer cells. While telomerase is essential for telomere maintenance in cells in vitro and in vivo, several studies now also indicate that telomerase may have telomere-independent functions. We previously showed that lowering the level of the telomerase ribonucleoprotein (RNP) enzyme complex causes rapid changes in cell cycle program in human and mouse cancer cells: partial reduction of telomerase induced by directly lowering telomerase RNP component levels causes altered transcriptional profiles and reduced glucose metabolism in human cancer cells, yet bulk telomere shortening or detectable telomere uncapping are not required for these effects. Work by others has shown that reduction of telomerase core protein component level reduced Wnt signaling and caused Wnt-mutant like developmental defects in Xenopus and mouse and aberrant hematopoiesis in zebrafish embryos. We acutely depleted telomerase RNA components in cultured T cells from healthy human adults using shRNAs targeting both the telomerase protein and the RNA component of telomerase (hTR), delivered immediately after in vitro stimulation. Cell proliferation and telomerase activity levels were quantified through the first and second stimulation cycles. Effects of such telomerase RNP reduction on cell proliferation were observed in time frames too short to be accounted for by bulk telomere attrition. Models to account for these findings will be discussed. Disclosures: Lin: Telome Health, Inc.: Consultancy, Equity Ownership. Blackburn:Telome Health, Inc: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

scholarly journals Telomere length and human hippocampal neurogenesis

2020 ◽  
Vol 45 (13) ◽  
pp. 2239-2247 ◽  
Author(s):  
Alish B. Palmos ◽  
Rodrigo R. R. Duarte ◽  
Demelza M. Smeeth ◽  
Erin C. Hedges ◽  
Douglas F. Nixon ◽  
...  
Keyword(s):  
Cognitive Function ◽  
Psychiatric Disorder ◽  
Telomere Length ◽  
Gene Networks ◽  
Hippocampal Neurogenesis ◽  
Telomere Maintenance ◽  
Adult Hippocampal Neurogenesis ◽  
Telomere Shortening ◽  
Age Related

Abstract Short telomere length is a risk factor for age-related disease, but it is also associated with reduced hippocampal volumes, age-related cognitive decline and psychiatric disorder risk. The current study explored whether telomere shortening might have an influence on cognitive function and psychiatric disorder pathophysiology, via its hypothesised effects on adult hippocampal neurogenesis. We modelled telomere shortening in human hippocampal progenitor cells in vitro using a serial passaging protocol that mimics the end-replication problem. Serially passaged progenitors demonstrated shorter telomeres (P ≤ 0.05), and reduced rates of cell proliferation (P ≤ 0.001), with no changes in the ability of cells to differentiate into neurons or glia. RNA-sequencing and gene-set enrichment analyses revealed an effect of cell ageing on gene networks related to neurogenesis, telomere maintenance, cell senescence and cytokine production. Downregulated transcripts in our model showed a significant overlap with genes regulating cognitive function (P ≤ 1 × 10−5), and risk for schizophrenia (P ≤ 1 × 10−10) and bipolar disorder (P ≤ 0.005). Collectively, our results suggest that telomere shortening could represent a mechanism that moderates the proliferative capacity of human hippocampal progenitors, which may subsequently impact on human cognitive function and psychiatric disorder pathophysiology.

scholarly journals G-Quadruplexes at Telomeres: Friend or Foe?

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3686 ◽  
Author(s):  
Tracy M. Bryan
Keyword(s):  
Tandem Repeats ◽  
Genome Instability ◽  
Protein Complexes ◽  
Telomere Maintenance ◽  
Telomeric Dna ◽  
Positive Role ◽  
Telomere Biology ◽  
Linear Chromosomes

Telomeres are DNA-protein complexes that cap and protect the ends of linear chromosomes. In almost all species, telomeric DNA has a G/C strand bias, and the short tandem repeats of the G-rich strand have the capacity to form into secondary structures in vitro, such as four-stranded G-quadruplexes. This has long prompted speculation that G-quadruplexes play a positive role in telomere biology, resulting in selection for G-rich tandem telomere repeats during evolution. There is some evidence that G-quadruplexes at telomeres may play a protective capping role, at least in yeast, and that they may positively affect telomere maintenance by either the enzyme telomerase or by recombination-based mechanisms. On the other hand, G-quadruplex formation in telomeric DNA, as elsewhere in the genome, can form an impediment to DNA replication and a source of genome instability. This review summarizes recent evidence for the in vivo existence of G-quadruplexes at telomeres, with a focus on human telomeres, and highlights some of the many unanswered questions regarding the location, form, and functions of these structures.

Lactobacillus inhibitor production against Escherichia coli and coaggregation ability with uropathogens

1988 ◽  
Vol 34 (3) ◽  
pp. 344-351 ◽  
Author(s):  
Gregor Reid ◽  
Jacqueline A. McGroarty ◽  
Rosanne Angotti ◽  
Roger L. Cook
Keyword(s):  
Escherichia Coli ◽  
Defense Mechanism ◽  
Inhibitory Effect ◽  
Second Phase ◽  
Vitro Assay ◽  
E Coli ◽  
Host Defense Mechanism ◽  
Important Host ◽  
Two Phases

Previous investigations have shown that certain strains of lactobacilli can competitively exclude uropathogens from attaching to uroepithelial cells and from causing urinary tract infection in animals. The finding of an inhibitory effect produced by Lactobacillus casei ssp. rhamnosus GR-1 against the growth of uropathogens was investigated further using two Escherichia coli indicator strains Hu 734 and ATCC 25922. There were two phases to the inhibitor studies. The first one using an agar sandwich technique showed that the inhibitor activity was heat stable and inhibitory to the E. coli. The second phase showed that MRS broth provided optimum lactobacilli growth and inhibitor production. In addition, the inhibition was present under conditions buffering for acid and pH. The data indicated that the inhibitory effect was not due to bacteriophages or hydrogen peroxide. Strain GR-1 was found to coaggregate with E. coli ATCC 25922 in urine, a phenomenon that has not previously been reported for urogenital bacteria. An in vitro assay system was developed to study the coaggregation of various lactobacilli and uropathogens. The results demonstrated that highest coaggregation scores occurred after 4 h incubation at 37 °C with lactobacilli and two type-1 fimbriated E. coli strains. Of the nine lactobacilli strains tested, each was found to coaggregate with 2 or more of the 13 uropathogens. The dominance of inhibitor-producing lactobacilli on the urogenital epithelium and the ability of these organisms to interact closely with uropathogens would constitute an important host defense mechanism against infection.

Telomerase Activity and Telomere Length of CD4+CD25+ Regulatory T-Cells under Conditions of In Vitro and In Vivo Expansion.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3857-3857
Author(s):  
Dominik G.F. Wolf ◽  
Anna M. Wolf ◽  
Christian Koppelstaetter ◽  
Holger F. Rumpold ◽  
Gert Mayer ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Telomere Length ◽  
Cancer Patients ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Healthy Human ◽  
Telomere Shortening

Abstract The expandability of CD4+CD25+ regulatory T-cells (Treg) has been shown in vitro and in vivo. Activation of telomerase activity is a prerequisite for clonal expansion and telomere maintenance in T-cells. There is currently no data available on the expression and function of telomerase in proliferating Treg. Analyses of telomere length by flow-FISH, real-time PCR and Southern blotting revealed that Treg isolated from healthy human volunteers have significantly shortened telomeres when compared to CD4+CD25− T-cells. However, telomere length is not further shortened in Treg isolated from the peripheral blood of cancer patients, despite the observation that the regulatory T-cell pool of these patients was significantly enlarged. To gain further insight into maintenance of telomere length of Treg, we induced in vitro proliferation of Treg by stimulation with anti-CD3 and IL-2. This led to a rapid increase of telomerase activity, as determined by PCR-ELISA. However, when we focused on the proliferating fraction of Treg using a sorting strategy based on the dilution of CFSE, we could show a significant telomere shortening in Treg with high proliferative and immmuno-suppressive capacity. Of note, proliferating CFSElow Treg are characterized by high telomerase activity, which however seems to be insufficient to avoid further telomere shortening under conditions of strong in vitro stimulation. In contrast, under conditions of in vivo expansion of Treg in cancer patients, the induction of telomerase activity is likely to compensate for further telomere erosion. These data might be of importance when considering the application of in vitro expanded Treg for the treatment of GvHD or autoimmune diseases, as telomere shortening might be associated with genomic instability.

Effective Telomerase Inhibition Via Expression of Dominant-Negative HTERT Leads to Altered Growth Kinetics, Elevated Apoptosis and Increased Radiosensitivity of Bcr-Abl-Positive K562 Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2935-2935
Author(s):  
Ute Brassat ◽  
Stefan Balabanov ◽  
Henning Wege ◽  
Daniel Roessler ◽  
Kerstin Borgmann ◽  
...  
Keyword(s):  
Growth Kinetics ◽  
K562 Cells ◽  
Telomerase Activity ◽  
Double Strand Breaks ◽  
Dominant Negative ◽  
Telomere Maintenance ◽  
Telomere Shortening ◽  
Telomerase Inhibition ◽  
Strand Breaks

Abstract Disease progression in CML is associated with accelerated telomere shortening caused by increased turnover of Bcr-Abl positive cells. We have recently proposed a model in which continuous telomere shortening is correlated with increasing genetic instability in Bcr-Abl-positive cells thus facilitating the acquisition of secondary genetic abnormalities and, as a consequence clonal succession and eventually clinical progression of the disease. Based on this hypothesis, telomerase upregulation in late stage disease is a prerequisite for prevention of replication-induced senescence in Bcr-Abl positive cells. As a consequence, treatment of CML with Telomerase inhibitors (TI) represents an attractive strategy aiming at the potential eradication of cycling CML stem cells. Therefore, we have exploited both pharmacological (small molecule inhibitor) and genetic strategies (dominant negative hTERT mutants, DN-hTERT) of telomerase inhibition in CML cells in vitro We first treated K562 cells with the pharmacological telomerase inhibitor BIBR1532 in vitro. After around 400 population doublings (PD), no differences in growth kinetics nor signs of senescence or apoptosis were observed in BIBR1532 treated cells despite of significant telomere shortening (22 base pairs (bp) per PD) compared to control cells. Furthermore, neither significant differences in mRNA expression of telomere/telomerase-associated proteins, nor accumulation of double strand breaks (DSBs) under irradiation was observed in treated cells with short telomeres as opposed to untreated control cells. The very slow shortening rate of 22bp/PD plus the lack of stigmata pointing to induction of senescence in K562 cells lead to the assumption that telomerase activity is not complete inhibited by the compound. In order to verify the potency of telomerase directed treatments in CML, we therefore expressed DN-hTERT in K562 cells. Integration of DN-hTERT led to a significant decrease in telomerase activity (measured by RQ-TRAP). Furthermore, DN-hTERT expressing cells underwent accelerated telomere shortening at a substantially higher rate (>100 bp/PD) from 15kb to around 4kb within 110 days of culture. In contrast to BIBR1532-treated cells, DN-hTERT expressing K562 cells slowed down growth kinetic in comparison to control cells after 80 days of culture. By using Annexin 5 staining, 25% of apoptotic cells could be detected in cells with critically short telomeres as compared to control cells (<3%). Finally, a significantly increased accumulation of double strand breaks (DSBs) detected by gammaH2AX foci after exposure to irradiation was observed in DN-hTERT K562 cells as compared to control cells pointing to an impaired DNA repair machinery in Bcr-Abl positive cells with disrupted telomere maintenance. In summary, the data suggest that pharmacological telomerase inhibition by BIBR1532 is insufficient to induce telomere-mediated senescence in Bcr-Abl-positive cells. However, accelerated telomere shortening, slowing down of growth kinetics, elevated apoptosis and increased radiosensitivity induced by expression of DN-hTERT indicate a therapeutic potential for telomerase-directed treatment strategies in CML.

scholarly journals The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time

2002 ◽  
Vol 157 (7) ◽  
pp. 1139-1149 ◽  
Author(s):  
Jordan W. Raff ◽  
Kim Jeffers ◽  
Jun-yong Huang
Keyword(s):  
Cell Cycle ◽  
Triple Point ◽  
Mitotic Spindle ◽  
Cyclin B ◽  
Second Phase ◽  
Point Mutant ◽  
Two Phases ◽  
Drosophila Cells

In Drosophila cells cyclin B is normally degraded in two phases: (a) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator; and (b) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. We show that the APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box–mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]–GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM–GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM–GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint.

Digestion in the tick,Argas persicus, Oken.

Parasitology ◽  
1964 ◽  
Vol 54 (3) ◽  
pp. 423-440 ◽  
Author(s):  
R. J. Tatchell
Keyword(s):  
Epithelial Cells ◽  
Waste Product ◽  
Aminopeptidase Activity ◽  
Second Phase ◽  
Rapid Phase ◽  
Argas Persicus ◽  
Intracellular Digestion ◽  
Two Phases

1.Studies on the histological changes during digestion in Argas persicus reveal that the ingestion of blood is accompanied by the destruction of the existing gut epithelial cells.2.The blood remains unlysed for 2–3 days while a new epithelium develops which contains cells that secrete a saliva-fast PAS-positive colloid that causes haemolysis.3.Other epithelial cells remove the freed erythrocytic nuclei by phagocytosis.4.Most of the gut cells then absorb protein from the lumen and intracellular digestion takes place leaving pure haematin granules as the waste product of the digestion of haemoglobin.5.After the initial rapid phase of digestion only relatively few cells show signs of absorptive and digestive activity.6.Absorption of protein is accompanied by increased alkaline phosphatase activity in the microvilli of the cell border; this activity is lost once absorption finishes and digestion begins.7.Strong aminopeptidase activity can be demonstrated at the border of some of the protein vacuoles.8.In vitro tests show that the gut proteinase is active only in the acid range with peaks at pH 2·6 and 3·8 and has a Km of 0·32 % with bovine serum albumin as substrate.9.In vivo data show that digestion, after haemolysis has occurred, takes place in two phases; the first is rapid and lasts approximately 1–2 weeks and is followed by the second phase which is slow and remains constant until the next blood feed.10.The proportion of the blood meal which remains after the rapid phase of digestion is determined by the sex and developmental stage of the tick and within each category it is constant and serves, in the absence of significant fat and glycogen reserves, as a food reserve.

scholarly journals Metabolism of free and esterified cholesterol by Leydig-cell tumour mitochondria

1973 ◽  
Vol 134 (2) ◽  
pp. 415-424 ◽  
Author(s):  
William R. Moyle ◽  
Robert L. Jungas ◽  
Roy O. Greep
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Leydig Cell ◽  
Bovine Serum ◽  
Incubation Temperature ◽  
Mitochondrial Fraction ◽  
Second Phase ◽  
Leydig Cell Tumour ◽  
Two Phases

1. Experiments were designed to localize intracellularly the enzymes and sterol substrates required for steroidogenesis in Leydig-cell tumours. Subcellular fractions were prepared by differential centrifugation of tumour homogenates. Both free and esterified cholesterol were associated primarily with the fractions sedimenting at 1400gav. and the lipid layer floating on the surface of the isolation tubes; they were not found in the mitochondria, where the conversion of cholesterol into pregnenolone occurred. 2. Hydrolysis of esterified cholesterol was required before it could be oxidized to pregnenolone. 3. An enzyme capable of hydrolysing cholesterol esters was located external to the mitochondria. 4. Mitochondria were subfractionated by allowing them to swell in 0.02m-phosphate buffer (pH7.2) and separating the inner and outer membranes by sedimentation in sucrose gradients. The outer membrane, identified by its content of monoamine oxidase, contained most of the cholesterol associated with the mitochondria. The inner membrane, identified by its content of succinate dehydrogenase, contained the cholesterol side-chain-cleaving enzyme and very little cholesterol. 5. Accumulation of sterols by the mitochondria was studied by incubating this fraction with labelled free and esterified cholesterol suspended in lipid-free bovine serum albumin. Two phases of cholesterol accumulation were observed. The first phase, requiring 10–15min, was independent of the incubation temperature, and was inhibited by the presence of bovine serum albumin in the incubation medium. The second phase of accumulation was independent of the serum albumin content of the medium but was inhibited by low incubation temperature. 6. Esterified cholesterol was not accumulated by the mitochondria after the initial rapid binding phase. 7. The findings suggest that cholesterol was not rapidly accumulated by the mitochondrial fraction in vitro and that mechanisms may be required to facilitate cholesterol transport into mitochondria in intact tumour cells during the periods in which steroidogenesis is stimulated maximally.

G-quadruplex unwinding helicases and their function in vivo

2017 ◽  
Vol 45 (5) ◽  
pp. 1173-1182 ◽  
Author(s):  
Markus Sauer ◽  
Katrin Paeschke
Keyword(s):  
Dna Replication ◽  
Genome Instability ◽  
Evolutionary Conservation ◽  
Telomere Maintenance ◽  
Cellular Functions ◽  
G Quadruplex

The concept that G-quadruplex (G4) structures can form within DNA or RNA in vitro has been long known and extensively discussed. In recent years, accumulating evidences imply that G-quadruplex structures form in vivo. Initially, inefficient regulation of G-quadruplex structures was mainly associated with genome instability. However, due to the location of G-quadruplex motifs and their evolutionary conservation, different cellular functions of these structures have been postulated (e.g. in telomere maintenance, DNA replication, transcription, and translation). Regardless of their function, efficient and controlled formation and unwinding are very important, because ‘mis’-regulated G-quadruplex structures are detrimental for a given process, causing genome instability and diseases. Several helicases have been shown to target and regulate specific G-quadruplex structures. This mini-review focuses on the biological consequences of G4 disruption by different helicases in vivo.

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