Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance

2020 ◽  
Vol 20 (6) ◽  
pp. 498-507 ◽  
Author(s):  
Connor A.H. Thompson ◽  
Judy M.Y. Wong
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Reverse Transcriptase ◽  
Cancer Cells ◽  
Telomere Length ◽  
Telomere Maintenance ◽  
Telomerase Reverse Transcriptase ◽  
Alternative Mechanism ◽  
Dependent Manner ◽  
Mitochondrial Integrity

Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase (TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed at higher levels than necessary for maintaining functional telomere length, suggesting other possible adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics, and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily show cellular protective effects, and nuclear TERT has been shown to protect against cell death following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria, TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding the spectrum of non-canonical functions of telomerase may have important implications for the rational design of anti-cancer chemotherapeutic drugs.

scholarly journals Evaluations of short telomere risk-associated single nucleotide polymorphisms in telomerase reverse transcriptase gene on telomere length maintenance

2019 ◽  
Author(s):  
Jialin Xu ◽  
Matthew A. Trudeau ◽  
Andrew J. Sandford ◽  
Judy M.Y. Wong
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Reverse Transcriptase ◽  
Telomere Length ◽  
Replicative Senescence ◽  
Telomere Maintenance ◽  
Telomerase Reverse Transcriptase ◽  
Nucleotide Polymorphisms ◽  
Primary Cells ◽  
Single Nucleotide ◽  
Telomere Biology

ABSTRACTTelomere biology disorders (TBDs) refer to a spectrum of tissue degenerative disorders caused by genetic mutations in telomere biology genes. Most patients with TBDs suffer from telomere maintenance defects secondary to telomerase deficiency. While the highly penetrant mutations in the telomerase reverse transcriptase (TERT) gene that drive disease onset and progression of TBDs are relatively rare, there exist several single nucleotide polymorphisms (SNPs) in TERT that have been linked to various diseases in the TBD spectrum. In this study, we investigated the biochemical properties of five TERT variants. In an ex vivo cell model, we found that primary human fibroblasts expressing nonsynonymous TERT SNPs had comparable cell growth kinetics to primary cells expressing WT-TERT, while a parallel vector control expressing-cell line entered replicative senescence. At the molecular level, primary cells expressing the minor alleles of two of the five TERT variants (A279T, ΔE441) had replication-dependent loss of telomere length. In an in vitro primer extension assay, these two variants showed reduced telomerase nucleotide addition processivity. Together, our data suggested that selective, common TERT variants could be revealed to harbour telomere maintenance defects, leading to a plausible explanation for their observed associations to telomere biology disorders.

Human Telomerase Reverse Transcriptase (hTERT) Deficiency in Myelodysplastic Syndrome (MDS) Demonstrates Mechanistic Linkage to Aplastic Anemia Pathophysiology

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 791-791
Author(s):  
Lili Yang ◽  
Adam W Mailloux ◽  
Dana E Rollison ◽  
Jong Park ◽  
Jeffrey S. Painter ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Reverse Transcriptase ◽  
Aplastic Anemia ◽  
Telomere Length ◽  
Telomerase Activity ◽  
S Phase ◽  
Telomere Maintenance ◽  
Telomerase Reverse Transcriptase ◽  
Htert Transcription

Abstract Abstract 791 Background: Myelodysplastic syndromes (MDS) are characterized by dysregulated myelopoiesis and peripheral cytopenias with enormous disease heterogeneity owing to diverse molecular pathobiology. The early manifestations of MDS, however, are relatively well conserved and include increased apoptosis coupled to excessive proliferation of myeloid progenitors. In addition to myeloid abnormalities, repertoire contraction and memory expansion is demonstrable in T cells. The notion that apoptosis of hematopoetic cells may be triggered through an immune–mediated mechanism arose from similarities with aplastic anemia (AA). Our recent data showed that MDS responsive to immunosuppressive therapy has accelerated naïve T cell turnover (ie, high proliferative index plus excessive cell death) which led us to hypothesize the presence of an inherent T cell abnormality impairing homeostatic regulation. AA can be caused by somatic mutations within telomere repair components. T-cells are one of a few somatic cells that retain telomerase function to control naïve T-cell survival, replication potential, and antigenic diversity. To this end, we examined telomere function and replicative burst capacity of MDS T cells as a possible mechanism for immune dysregulation. Methods: Primary specimens from MDS (n=37), AA (n=8), and controls (n=42) were investigated. Peripheral blood mononuclear cells were isolated from patient blood or buffy coats by Ficoll-Hypaque gradient centrifugation. Purified CD3+ T cells were isolated using negative selection and then stimulated with anti-CD3/anti-CD28 T cell activator beads (Dynabead®) for 3 days. Telomere length was assessed by quantitative PCR (q-PCR) and telomerase enzymatic function measured by Telomere Repeat Amplification Protocol (TRAP) assays. Results: Mean telomere length in purified T cells was significantly shorter among MDS patients compared to controls after adjusting for age and sex (p<0.0001). To assess telomerase repair function in MDS T-cells, we performed TRAP assays with purified T cells after stimulation and found that inducible telomerase activity is severely suppressed in MDS compare to controls. In comparison to controls, the inducible telomerase activity fell below the 95% confidence internal in all cases (MDS median 18.70, 95% CI, 15.93–20.54 vs control median 45.0, 95% CI, 45.79 – 64.5, p<0.0001) and the amount of telomerase activity was unrelated to risk stratification by the International Prognostic Scoring System (IPSS), World Health Organization (WHO) classification, and age indicating that it is a frequent abnormality in the disease. Analysis of telomerase function and telomere length in T cells from patients with AA showed a similar deficiency in telomerase repair function. The mechanism responsible for telomerase insufficiency in MDS was mediated by defective induction of telomerase reverse transcriptase (hTERT) transcription; the key enzyme involved in telomere maintenance. Next, to determine the functional consequences of the disturbance in telomere repair in MDS, the ability of T cells to enter S-phase and to undergo an antigen-induced proliferative burst were examined. TCR signaling was shown to be preserved, evidenced by induction of an early activation antigen CD69. Although some cells were capable of entering S-phase, the replicative burst potential was severely impaired in T cells form all patients. Telomere repair is exclusively present in naïve T cells and progressively declines after memory transition. TCR triggered telomerase activity was measured in sorted naïve (CD45RA+, CD45RO-) and memory (CD45RO+, CD45RA-) T cells. The telomere length in naïve cells was shorter in MDS patients compared to controls (p=0.018) and the telomerase activity was suppressed in naïve MDS T cells (p=0.0207) indicating that telomere dysfunction underlies the altered homeostasis of naïve T cells in MDS, a feature mechanistically akin to AA and other telomere repair disorders. Conclusion: Results of this study indicate that there is loss of telomere maintenance in naïve T cells due to a defect in hTERT transcription is associated with impaired replicative potential. This abnormality in naïve T cell homeostasis represents an inherent defect that contributes to a memory cell growth advantage and repertoire contraction associated with autoimmunity in AA and MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Withaferin A Triggers Apoptosis and DNA Damage in Bladder Cancer J82 Cells through Oxidative Stress

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1063
Author(s):  
Tsu-Ming Chien ◽  
Kuang-Han Wu ◽  
Ya-Ting Chuang ◽  
Yun-Chiao Yeh ◽  
Hui-Ru Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Bladder Cancer ◽  
Cancer Cells ◽  
Withaferin A ◽  
Heme Oxygenase 1 ◽  
Dependent Manner ◽  
Dna Damages ◽  
Bladder Cancer Cells ◽  
Stress Dependent

Withaferin A (WFA), the Indian ginseng bioactive compound, exhibits an antiproliferation effect on several kinds of cancer, but it was rarely reported in bladder cancer cells. This study aims to assess the anticancer effect and mechanism of WFA in bladder cancer cells. WFA shows antiproliferation to bladder cancer J82 cells based on the finding of the MTS assay. WFA disturbs cell cycle progression associated with subG1 accumulation in J82 cells. Furthermore, WFA triggers apoptosis as determined by flow cytometry assays using annexin V/7-aminoactinomycin D and pancaspase detection. Western blotting also supports WFA-induced apoptosis by increasing cleavage of caspases 3, 8, and 9 and poly ADP-ribose polymerase. Mechanistically, WFA triggers oxidative stress-association changes, such as the generation of reactive oxygen species and mitochondrial superoxide and diminishment of the mitochondrial membrane potential, in J82 cells. In response to oxidative stresses, mRNA for antioxidant signaling, such as nuclear factor erythroid 2-like 2 (NFE2L2), catalase (CAT), superoxide dismutase 1 (SOD1), thioredoxin (TXN), glutathione-disulfide reductase (GSR), quinone dehydrogenase 1 (NQO1), and heme oxygenase 1 (HMOX1), are overexpressed in J82 cells. In addition, WFA causes DNA strand breaks and oxidative DNA damages. Moreover, the ROS scavenger N-acetylcysteine reverts all tested WFA-modulating effects. In conclusion, WFA possesses anti-bladder cancer effects by inducing antiproliferation, apoptosis, and DNA damage in an oxidative stress-dependent manner.

scholarly journals Expression of Telomerase RNA Template, but Not Telomerase Reverse Transcriptase, Is Limiting for Telomere Length Maintenance In Vivo

2004 ◽  
Vol 24 (16) ◽  
pp. 7024-7031 ◽  
Author(s):  
Y. Jeffrey Chiang ◽  
Michael T. Hemann ◽  
Karen S. Hathcock ◽  
Lino Tessarollo ◽  
Lionel Feigenbaum ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Telomere Length ◽  
Telomerase Activity ◽  
Telomere Maintenance ◽  
Gene Copy ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Wild Type ◽  
Telomere Elongation

ABSTRACT Telomerase consists of two essential components, the telomerase RNA template (TR) and telomerase reverse transcriptase (TERT). The haplo-insufficiency of TR was recently shown to cause one form of human dyskeratosis congenita, an inherited disease marked by abnormal telomere shortening. Consistent with this finding, we recently reported that mice heterozygous for inactivation of mouse TR exhibit a similar haplo-insufficiency and are deficient in the ability to elongate telomeres in vivo. To further assess the genetic regulation of telomerase activity, we have compared the abilities of TR-deficient and TERT-deficient mice to maintain or elongate telomeres in interspecies crosses. Homozygous TERT knockout mice had no telomerase activity and failed to maintain telomere length. In contrast, TERT+/− heterozygotes had no detectable defect in telomere elongation compared to wild-type controls, whereas TR+/− heterozygotes were deficient in telomere elongation. Levels of TERT mRNA in heterozygous mice were one-third to one-half the levels expressed in wild-type mice, similar to the reductions in telomerase RNA observed in TR heterozygotes. These findings indicate that both TR and TERT are essential for telomere maintenance and elongation but that gene copy number and transcriptional regulation of TR, but not TERT, are limiting for telomerase activity under the in vivo conditions analyzed.

scholarly journals Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

2016 ◽  
Vol 37 (3) ◽  
Author(s):  
Kunitoshi Chiba ◽  
Jacob M. Vogan ◽  
Robert A. Wu ◽  
Manraj S. Gill ◽  
Xiaozhu Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Reverse Transcriptase ◽  
Cancer Cells ◽  
Genetically Engineered ◽  
Telomere Maintenance ◽  
Proliferative Potential ◽  
Telomerase Reverse Transcriptase ◽  
Endogenous Expression ◽  
Human Telomerase

ABSTRACT Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo.

scholarly journals Telomerase Reverse Transcriptase Is Required for the Localization of Telomerase RNA to Cajal Bodies and Telomeres in Human Cancer Cells

2008 ◽  
Vol 19 (9) ◽  
pp. 3793-3800 ◽  
Author(s):  
Rebecca L. Tomlinson ◽  
Eladio B. Abreu ◽  
Tania Ziegler ◽  
Hinh Ly ◽  
Christopher M. Counter ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Cancer Cells ◽  
Human Cancer ◽  
Telomere Maintenance ◽  
Cajal Body ◽  
Cajal Bodies ◽  
Telomerase Reverse Transcriptase ◽  
Telomerase Rna ◽  
Human Cancer Cells ◽  
Essential Components

Telomere maintenance by telomerase is critical for the unlimited division potential of most human cancer cells. The two essential components of human telomerase, telomerase RNA (hTR) and telomerase reverse transcriptase (hTERT), are recruited from distinct subnuclear sites to telomeres during S phase. Throughout the remainder of the cell cycle hTR is found primarily in Cajal bodies. The localization of hTR to Cajal bodies and telomeres is specific to cancer cells where telomerase is active and is not observed in primary cells. Here we show that the trafficking of hTR to both telomeres and Cajal bodies depends on hTERT. RNA interference–mediated depletion of hTERT in cancer cells leads to loss of hTR from both Cajal bodies and telomeres without affecting hTR levels. In addition, expression of hTERT in telomerase-negative cells (including primary and ALT cancer cell lines) induces hTR to localize to both sites. Factors that did not stimulate hTR localization in our experiments include increased hTR RNA levels and Cajal body numbers, and expression of SV40 large T antigen and oncogenic Ras. Our findings suggest that the trafficking of telomerase to Cajal bodies and telomeres in cancer cells correlates with and depends on the assembly of the enzyme.

scholarly journals BRD4 inhibitors block telomere elongation

2017 ◽  
Author(s):  
Steven Wang ◽  
Alexandra M. Pike ◽  
Stella S. Lee ◽  
Carla J. Connelly ◽  
Carol W. Greider
Keyword(s):  
Cancer Cells ◽  
Telomere Length ◽  
Term Treatment ◽  
Telomere Maintenance ◽  
Dependent Manner ◽  
Telomere Elongation ◽  
Long Term Treatment ◽  
Brd4 Inhibition ◽  
Shrna Screen

ABSTRACTCancer cells maintain telomere length equilibrium to avoid senescence and apoptosis induced by short telomeres, which are triggered by the DNA damage response. Limiting the potential for telomere maintenance in cancer cells has been long been proposed as a therapeutic target. Using an unbiased shRNA screen targeting known kinases, we identified bromodomain 4 (BRD4) as a telomere length regulator. Four independent BRD4 inhibitors blocked telomere elongation, in a dose dependent manner, in mouse cells overexpressing telomerase. Long-term treatment with BRD4 inhibitors caused telomere shortening in both mouse and human cells, suggesting BRD4 plays a role in telomere maintenancein vivo. Telomerase enzymatic activity was not directly affected by BRD4 inhibition. BRD4 is in clinical trials for a number of cancers, but its effects on telomere maintenance have not been previously reported investigated.

scholarly journals Vernonia calvoana Shows Promise towards the Treatment of Ovarian Cancer

2020 ◽  
Vol 21 (12) ◽  
pp. 4429
Author(s):  
Ariane T. Mbemi ◽  
Jennifer N. Sims ◽  
Clement G. Yedjou ◽  
Felicite K. Noubissi ◽  
Christian R. Gomez ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Ovarian Cancer ◽  
Dna Damage ◽  
Antitumor Activity ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Ovarian Cancer Cells ◽  
Dependent Manner ◽  
Cycle Arrest

The treatment for ovarian cancers includes chemotherapies which use drugs such as cisplatin, paclitaxel, carboplatin, platinum, taxanes, or their combination, and other molecular target therapies. However, these current therapies are often accompanied with side effects. Vernonia calvoana (VC) is a valuable edible medicinal plant that is widespread in West Africa. In vitro data in our lab demonstrated that VC crude extract inhibits human ovarian cancer cells in a dose-dependent manner, suggesting its antitumor activity. From the VC crude extract, we have generated 10 fractions and VC fraction 7 (F7) appears to show the highest antitumor activity towards ovarian cancer cells. However, the mechanisms by which VC F7 exerts its antitumor activity in cancer cells remain largely unknown. We hypothesized that VC F7 inhibits cell proliferation and induces DNA damage and cell cycle arrest in ovarian cells through oxidative stress. To test our hypothesis, we extracted and fractionated VC leaves. The effects of VC F7 were tested in OVCAR-3 cells. Viability was assessed by the means of MTS assay. Cell morphology was analyzed by acridine orange and propidium iodide (AO/PI) dye using a fluorescent microscope. Oxidative stress biomarkers were evaluated by the means of lipid peroxidation, catalase, and glutathione peroxidase assays, respectively. The degree of DNA damage was assessed by comet assay. Cell cycle distribution was assessed by flow cytometry. Data generated from the MTS assay demonstrated that VC F7 inhibits the growth of OVCAR-3 cells in a dose-dependent manner, showing a gradual increase in the loss of viability in VC F7-treated cells. Data obtained from the AO/PI dye assessment revealed morphological alterations and exhibited characteristics such as loss of cellular membrane integrity, cell shrinkage, cell membrane damage, organelle breakdown, and detachment from the culture plate. We observed a significant increase (p < 0.05) in the levels of malondialdhyde (MDA) production in treated cells compared to the control. A gradual decrease in both catalase and glutathione peroxidase activities were observed in the treated cells compared to the control. Data obtained from the comet assay showed a significant increase (p < 0.05) in the percentages of DNA cleavage and comet tail length. The results of the flow cytometry analysis indicated VC F7 treatment caused cell cycle arrest at the S-phase checkpoint. Taken together, our results demonstrate that VC F7 exerts its anticancer activity by inhibiting cell proliferation, inducing DNA damage, and causing cell cycle arrest through oxidative stress in OVAR-3 cells. This finding suggests that VC F7 may be a potential alternative dietary agent for the prevention and/or treatment of ovarian cancer.

Regulation And Effect Of Telomerase And Telomeric Length In Stem Cells

2020 ◽  
Vol 15 ◽  
Author(s):  
Basak Celtikci ◽  
Gulnihal Kulaksiz Erkmen ◽  
Zeliha Gunnur Dikmen
Keyword(s):  
Telomere Length ◽  
Somatic Cells ◽  
Telomere Maintenance ◽  
The Other ◽  
Telomerase Reverse Transcriptase ◽  
Human Telomerase Reverse Transcriptase ◽  
Telomere Shortening ◽  
Maintenance Mechanism ◽  
Associated Diseases ◽  
Human Telomerase

: Telomeres are the protective end caps of eukaryotic chromosomes and they decide the proliferative lifespan of somatic cells, as the guardians of the cell replication. Telomere length in leucocytes reflects telomere length in other somatic cells. Leucocyte telomere length can be a biomarker of human ageing. The risk of diseases, which are associated with reduced cell proliferation and tissue degeneration, including aging or aging-associated diseases, such as dyskeratosis congenita, cardiovascular diseases, pulmonary fibrosis and aplastic anemia, are correlated with an increase in short telomeres. On the other hand, the risk of diseases, which are associated with increased proliferative growth, including major cancers, is correlated with long telomeres. In most of the cancers, a telomere maintenance mechanism during DNA replication is essential. The reactivation of the functional ribonucleoprotein holoenzyme complex [telomerase] starts the cascade from normal and premalignant somatic cells to advanced malignant cells. Telomerase is overexpressed during the development of cancer and embryonic stem cells, through controlling genome integrity, cancer formation and stemness. Cancer cells have mechanisms to maintain telomeres to avoid initiation of cellular senescence or apoptosis, and halting cell division by critically short telomeres. Modulation of the human telomerase reverse transcriptase is the ratelimiting step for the production of functional telomerase and the telomere maintenance. Human telomerase reverse transcriptase promoter promotes its gene expression only in tumor cells, but not in normal cells. Some cancers activate an alternative lengthening of telomeres maintenance mechanism via DNA recombination to unshorten their telomeres. Not only heritability but also oxidative stress, inflammation, environmental factors, and therapeutic interventions have an effect on telomere shortening, explaining the variability in telomere length across individuals. There have been a large number of publications, which correlate human diseases with progressive telomere shortening. Telomere length of an individual at birth is also important to follow up telomere shortening, and it can be used as biomarkers for healthy aging. On the other hand, understanding of cellular stress factors, which affect stem cell behavior, will be useful in regeneration or treatment in cancer and age-associated diseases. In this review, we will understand the connection between stem cell and telomere biology, cancer, and aging-associated diseases. This connection may be useful for discovering novel drug targets and improve outcomes for patients having cancer and aging-associated diseases.

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