Telomerase inhibition on acute myeloid leukemia stem cell induced apoptosis with both intrinsic and extrinsic pathways.

AIMS: Acute Myeloid Leukemia (AML) is an invasive and lethal blood cancer caused by a rare population of Leukemia Stem Cells (LSCs). Telomerase activation is a limitless self-renewal process in LSCs. Apart from telomerase role in telomere lengthening, telomerase (especially hTERT subunit) inhibits intrinsic-, extrinsic-, and p53- mediated apoptosis pathways. In this study, the effect of Telomerase Inhibition (TI) on intrinsic-, extrinsic-, p53-mediated apoptosis, and DNMT3a and TET epigenetic markers in stem (CD34+) and differentiated (CD34-) AML cells is evaluated. MAIN METHODS: High-purity CD34+ (primary AML and KG-1a) cells were enriched using the Magnetic-Activated Cell Sorting (MACS) system. CD34+ and CD34- (primary AML and KG-1a) cells were treated with BIBR1532 and then, MTT assay, Annexin V/7AAD, Ki-67 assay, Telomere Length (TL) measurement, and transcriptional alterations of p53, hTERT, TET2, DNMT3a were analyzed. Finally, apoptosis-related genes and proteins were studied. KEY FINDINGS: TI with the IC50 values of 83.5, 33.2, 54.3, and 24.6 µM in CD34+ and CD34- (primary AML and KG-1a) cells significantly inhibited cell proliferation and induced apoptosis. However, TI had no significant effect on TL. The results also suggested TI induced intrinsic-, extrinsic-, and p53-mediated apoptosis. It was shown that the expression levels of DNMT3a and TET2 epigenetic markers were highly increased following TI. SIGNIFICANCE: In total, it was revealed that TI induced apoptosis through intrinsic, extrinsic, and p53 pathways and increased the expression of DNMT3a and TET2 epigenetic markers.

The dual distinct role of telomerase in repression of senescence and myofibroblast differentiation.

Many aging related diseases such as cancer implicate the myofibroblast in disease progression. Furthermore genesis of the myofibroblast is associated with manifestation of cellular senescence of unclear significance. In this study we investigated the role of a common regulator, namely telomerase reverse transcriptase (TERT), in order to evaluate the potential significance of this association between both processes. We analyzed the effects of TERT overexpression or deficiency on expression of CDKN2A and ACTA2 as indicators of senescence and differentiation, respectively. We assess binding of TERT or YB-1, a repressor of both genes, to their promoters. TERT repressed both CDKN2A and ACTA2 expression, and abolished stress-induced expression of both genes. Conversely, TERT deficiency enhanced their expression. Altering CDKN2A expression had no effect on ACTA2 expression. Both TERT and YB-1 were shown to bind the CDKN2A promoter but only YB-1 was shown to bind the ACTA2 promoter. TERT overexpression inhibited CDKN2A promoter activity while stimulating YB-1 expression and activation to repress ACTA2 gene. TERT repressed myofibroblast differentiation and senescence via distinct mechanisms. The latter was associated with TERT binding to the CDKN2A promoter, but not to the ACTA2 promoter, which may require interaction with co-factors such as YB-1.

Potential of Telomerase in Age-Related Macular Degeneration-Involvement of Senescence, DNA Damage Response and Autophagy and a Key Role of PGC-1α.

Age-related macular degeneration (AMD), the main cause of vision loss in the elderly, is associated with oxidation in the retina cells promoting telomere attrition. Activation of telomerase was reported to improve macular functions in AMD patients. The catalytic subunit of human telomerase (hTERT) may directly interact with proteins important for senescence, DNA damage response, and autophagy, which are impaired in AMD. hTERT interaction with mTORC1 (mTOR (mechanistic target of rapamycin) complex 1) and PINK1 (PTEN-induced kinase 1) activates macroautophagy and mitophagy, respectively, and removes cellular debris accumulated over AMD progression. Ectopic expression of telomerase in retinal pigment epithelium (RPE) cells lengthened telomeres, reduced senescence, and extended their lifespan. These effects provide evidence for the potential of telomerase in AMD therapy. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) may be involved in AMD pathogenesis through decreasing oxidative stress and senescence, regulation of vascular endothelial growth factor (VEGF), and improving autophagy. PGC-1α and TERT form an inhibitory positive feedback loop. In conclusion, telomerase activation and its ectopic expression in RPE cells, as well as controlled clinical trials on the effects of telomerase activation in AMD patients, are justified and should be assisted by PGC-1α modulators to increase the therapeutic potential of telomerase in AMD.

Telomerase: a key player in the pathogenesis of non-alcoholic fatty liver disease?

Introduction: Telomerase is a basic nuclear protein reverse transcriptase, which plays a key role in maintaining telomere stability, genome integrity, long-term cell activity, and potential continued proliferation.Area covered: This narrative review discusses key research advances involving telomerase in the development and progression of nonalcoholic fatty liver disease (NAFLD). The review evaluates 9a) whether the assessment of telomerase can be used as a noninvasive diagnostic tool; and (b) whether modification of telomerase function might be a useful potential therapeutic target for treatment of NAFLD. Furthermore, the relationship between telomerase and other chronic metabolic diseases is evaluated.Expert opinion: Several experimental and preclinical studies have suggested that telomerase plays an important role in the development of NAFLD. However, further mechanistic studies are needed to prove a causal relationship and to better elucidate whether the measurement of telomerase has utility as a diagnostic tool or whether pharmacological manipulation of telomerase has therapeutic potential in NAFLD treatment.

Composition and Function of Telomerase-A Polymerase Associated with the Origin of Eukaryotes.

The canonical DNA polymerases involved in the replication of the genome are unable to fully replicate the physical ends of linear chromosomes, called telomeres. Chromosomal termini thus become shortened in each cell cycle. The maintenance of telomeres requires telomerase-a specific RNA-dependent DNA polymerase enzyme complex that carries its own RNA template and adds telomeric repeats to the ends of chromosomes using a reverse transcription mechanism. Both core subunits of telomerase-its catalytic telomerase reverse transcriptase (TERT) subunit and telomerase RNA (TR) component-were identified in quick succession in Tetrahymena more than 30 years ago. Since then, both telomerase subunits have been described in various organisms including yeasts, mammals, birds, reptiles and fish. Despite the fact that telomerase activity in plants was described 25 years ago and the TERT subunit four years later, a genuine plant TR has only recently been identified by our group. In this review, we focus on the structure, composition and function of telomerases. In addition, we discuss the origin and phylogenetic divergence of this unique RNA-dependent DNA polymerase as a witness of early eukaryotic evolution. Specifically, we discuss the latest information regarding the recently discovered TR component in plants, its conservation and its structural features.

The secrets of telomerase: Retrospective analysis and future prospects.

Telomerase plays a significant role to maintain and regulate the telomere length, cellular immortality and senescence by the addition of guanine-rich repetitive sequences. Chronic inflammation or oxidative stress-induced infection downregulates TERT gene modifying telomerase activity thus contributing to the early steps of gastric carcinogenesis process. Furthermore, telomere-telomerase system performs fundamental role in the pathogenesis and progression of diabetes mellitus as well as in its vascular intricacy. The cessation of cell proliferation in cultured cells by inhibiting the telomerase activity of transformed cells renders the rationale for culling of telomerase as a target therapy for the treatment of metabolic disorders and various types of cancers. In this article, we have briefly described the role of immune system and malignant cells in the expression of telomerase with critical analysis on the gaps and potential for future studies. The key findings regarding the secrets of the telomerase summarized in this article will help in future treatment modalities for the prevention of various types of cancers and metabolic disorders notably diabetes mellitus.

[Healthy Aging: Antioxidants, Uncouplers and/or Telomerase?]

The free radical theory of aging was proposed in 1956. Although it does not fully describe the mechanisms of aging, it is generally accepted that reactive oxygen species (ROS) are one of the pathogenetic factors in aging and, in particular, in the development of pathologies associated with aging. The main source of ROS in the cell is mitochondria. Antioxidants directed to mitochondria have a positive effect, but have low efficiency. The problem is that increased amounts of antioxidants disrupt normal cellular redox reactions, and a low amount of antioxidants is not able to seriously affect the processes. Protection against ROS may be more effective if the rate of ROS formation is reduced. There is a natural mitochondrial uncoupling process that significantly reduces ROS production. The weak uncoupler dinitrophenol (DNP) prolongs the life span of mice, reduces traumatic brain damage, and inhibits the development of a number of neurodegenerative diseases. Unfortunately, DNP has a number of disadvantages that hinder its practical use. Uncoupling of oxidative phosphorylation by free fatty acids is a natural mechanism, the activation of which can be used in medicine. The third (after antioxidants and uncouplers), but so far little studied, method of reducing ROS is telomerase, which, under conditions of oxidative stress, is transported into the mitochondria and improves cell survival by reducing ROS production.

Alternative Lengthening of Telomeres: Building Bridges To Connect Chromosome Ends.

Alternative lengthening of telomeres (ALT) is a mechanism of telomere maintenance that is observed in many of the most recalcitrant cancer subtypes. Telomeres in ALT cancer cells exhibit a distinctive nucleoprotein architecture shaped by the mismanagement of chromatin that fosters cycles of DNA damage and replicative stress that activate homology-directed repair (HDR). Mutations in specific chromatin-remodeling factors appear to be key determinants of the emergence and survival of ALT cancer cells. However, these may represent vulnerabilities for the targeted elimination of ALT cancer cells that infiltrate tissues and organs to become devastating tumors. In this review we examine recent findings that provide new insights into the factors and mechanisms that mediate telomere length maintenance and survival of ALT cancer cells.

FLASH Irradiation Spares Lung Progenitor Cells and Limits the Incidence of Radio-induced Senescence.

PURPOSE: One of the main limitations to anticancer radiotherapy lies in irreversible damage to healthy tissues located within the radiation field. "FLASH" irradiation at very high dose-rate is a new treatment modality that has been reported to specifically spare normal tissue from late radiation-induced toxicity in animal models and therefore could be a promising strategy to reduce treatment toxicity. EXPERIMENTAL DESIGN: Lung responses to FLASH irradiation were investigated by qPCR, single-cell RNA sequencing (sc-RNA-Seq), and histologic methods during the acute wound healing phase as well as at late stages using C57BL/6J wild-type and Terc-/- mice exposed to bilateral thorax irradiation as well as human lung cells grown in vitro. RESULTS: In vitro studies gave evidence of a reduced level of DNA damage and induced lethality at the advantage of FLASH. In mouse lung, sc-RNA-seq and the monitoring of proliferating cells revealed that FLASH minimized the induction of proinflammatory genes and reduced the proliferation of progenitor cells after injury. At late stages, FLASH-irradiated lungs presented less persistent DNA damage and senescent cells than after CONV exposure, suggesting a higher potential for lung regeneration with FLASH. Consistent with this hypothesis, the beneficial effect of FLASH was lost in Terc-/- mice harboring critically short telomeres and lack of telomerase activity. CONCLUSIONS: The results suggest that, compared with conventional radiotherapy, FLASH minimizes DNA damage in normal cells, spares lung progenitor cells from excessive damage, and reduces the risk of replicative senescence.

Role of telomerase in the tumour microenvironment.

Telomeres are specialized genomic structures that protect chromosomal ends to maintain genomic stability. Telomeric length is primarily regulated by the telomerase complex, essentially consisting of an RNA template (TERC), an enzymatic subunit (telomerase reverse transcriptase, TERT). In humans, telomerase activity is repressed during embryonic differentiation and is absent in most somatic cells. However, it is upregulated or reactivated in 80%-90% of the primary tumours in humans. The human TERT (hTERT) plays a pivotal role in cellular immortality and tumourigenesis. However, the molecular mechanisms of telomerase functioning in cancer have not been fully understood beyond the telomere maintenance. Several research groups, including ours, have demonstrated that hTERT possesses vital functions independent of its telomere maintenance, including angiogenesis, inflammation, cancer cell stemness, and epithelial-mesenchymal transformation (EMT). All these telomere-independent activities of hTERT may contribute to the regulation of the dynamics and homeostasis of the tumour microenvironment (TME), thereby promoting tumour growth and development. Cancer progression and metastasis largely depend upon the interactions between cancer cells and their microenvironment. In this review, the involvement of TERT in the tumour microenvironment and the underlying implications in cancer therapeutics have been summarized.

The Tumor-Immune Response Is Not Compromised by Mesenchymal Stromal Cells in Humanized Mice.

Therapeutic uses of mesenchymal stromal cells (MSCs) have emerged over the past decade. Yet, their effect on tumor growth remains highly debated, particularly in an immune competent environment. In this study, we wanted to investigate the impact of human umbilical cord-derived MSCs (hUC-MSCs) on tumor growth in humanized mice generated by the human adoptive transfer of PBMCs or the cotransplantation of hematopoietic stem cells and human thymic tissue (human BLT [Hu-BLT]). Our results showed that the growth and immune rejection of engineered human fibroblastic tumors was not altered by the injection of hUC-MSCs in immune-deficient or humanized mice, respectively. This was observed whether tumor cells were injected s.c. or i.v. and independently of the injection route of the hUC-MSCs. Moreover, only in Hu-BLT mice did hUC-MSCs have some effects on the tumor-immune infiltrate, yet without altering tumor growth. These results demonstrate that hUC-MSCs do not promote fibroblastic tumor growth and neither do they prevent tumor infiltration and rejection by immune cells in humanized mice.

SMUG1 Promotes Telomere Maintenance through Telomerase RNA Processing.

Telomerase biogenesis is a complex process where several steps remain poorly understood. Single-strand-selective uracil-DNA glycosylase (SMUG1) associates with the DKC1-containing H/ACA ribonucleoprotein complex, which is essential for telomerase biogenesis. Herein, we show that SMUG1 interacts with the telomeric RNA component (hTERC) and is required for co-transcriptional processing of the nascent transcript into mature hTERC. We demonstrate that SMUG1 regulates the presence of base modifications in hTERC, in a region between the CR4/CR5 domain and the H box. Increased levels of hTERC base modifications are accompanied by reduced DKC1 binding. Loss of SMUG1 leads to an imbalance between mature hTERC and its processing intermediates, leading to the accumulation of 3'-polyadenylated and 3'-extended intermediates that are degraded in an EXOSC10-independent RNA degradation pathway. Consequently, SMUG1-deprived cells exhibit telomerase deficiency, leading to impaired bone marrow proliferation in Smug1-knockout mice.

Phage N15 protelomerase resolves its tos recognition site into hairpin telomeres within mammalian cells.

Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human ß-globin gene expressed for at least 120 h in non-ß-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.

Raising the bar in anticancer therapy: recent advances in, and perspectives on, telomerase inhibitors.

Telomerase is a ribonucleic reverse transcriptase enzyme that uses an integral RNA component as a template to add tandem telomeric DNA repeats, TTAGGG, at the 3' end of the chromosomes. 85-90% of human tumors and their derived cell lines predominantly express high levels of telomerase, therefore contributing to cancer cell development. However, in normal cells, telomerase activity is almost always absent except in germ cells and stem cells. This differential expression has been exploited to develop highly specific and potent cancer therapeutics. In this review, we outline recent advances in the development of telomerase inhibitors as anticancer agents.

BPDE and B[a]P induce mitochondrial compromise by ROS-mediated suppression of the SIRT1/TERT/PGC-1α pathway in spermatogenic cells both in vitro and in vivo.

There is increasing evidence that indicates benzo[a]pyrene (B[a]P) and its active metabolite benzo[a]pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) are endocrine disruptors that can cause reproductive toxicity. Nevertheless, the underlying mechanisms are still obscure. The present study investigates the impacts of B[a]P and BPDE on mitochondria, a sensitive target affected by multiple chemicals, in spermatogenic cells. It showed that BPDE treatment induced mitochondrial dysfunction and the inhibition of mitochondrial biogenesis in mouse spermatocyte-derived cells (GC-2). These effects were efficiently mitigated by pretreatment with ZLN005, an activator of PGC-1α, in GC-2 cells. TERT knockdown and re-expression cell models were established to demonstrate that TERT regulated the BPDE-induced mitochondrial damage via PGC-1α signaling in GC-2 cells. Moreover, upregulating or knockdown SIRT1 expression attenuated or aggravated BPDE-induced mitochondrial compromise by activating or inhibiting, respectively, the TERT and PGC-1α molecules in GC-2 cells. Finally, we observed that BPDE markedly elevated oxidative stress in GC-2 cells. Resveratrol and N-acetylcysteine, as reactive oxygen species (ROS) scavengers, attenuated BPDE-mediated mitochondrial damage by increasing SIRT1 activity and expression in GC-2 cells. The in vitro results were corroborated by in vivo experiments in rats treated with B[a]P for 4 weeks. B[a]P administration caused mitochondrial damage and mitochondria-dependent apoptosis in spermatogenic cells, as well as the decreased expression of SIRT1, TERT, and PGC-1α. In summary, the results of the present study demonstrate that B[a]P and BPDE induce mitochondrial damage through ROS production that suppresses SIRT1/TERT/PGC-1a signaling and mediate B[a]P- and BPDE-mediated reproductive toxicity.

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

How RNAi machinery enters the world of telomerase.

Human telomerase holoenzyme consists of the catalytic component TERT and the template RNA TERC. However, a network of accessory proteins plays key roles in its assembly, localization and stability. Defects in genes involved in telomerase biology affect the renewal of critical stem cell populations and cause disorders such as telomeropathies. Moreover, activation of telomerase in somatic cells allows neoplastic cells to proliferate indefinitely, thus contributing to tumorigenesis. For these reasons, identification of new players involved in telomerase regulation is crucial for the determination of novel therapeutic targets and biomarkers. In the very last years, increasing evidence describes components of the RNAi machinery as a new layer of complexity in human telomerase activity. In this review, we will discuss how AGO2 and other proteins which collaborate with AGO2 in RNAi pathway play a pivotal role in TERC stability and function.

Length-independent telomere damage drives post-mitotic cardiomyocyte senescence.

Ageing is the biggest risk factor for cardiovascular disease. Cellular senescence, a process driven in part by telomere shortening, has been implicated in age-related tissue dysfunction. Here, we address the question of how senescence is induced in rarely dividing/post-mitotic cardiomyocytes and investigate whether clearance of senescent cells attenuates age-related cardiac dysfunction. During ageing, human and murine cardiomyocytes acquire a senescent-like phenotype characterised by persistent DNA damage at telomere regions that can be driven by mitochondrial dysfunction and crucially can occur independently of cell division and telomere length. Length-independent telomere damage in cardiomyocytes activates the classical senescence-inducing pathways, p21CIP and p16INK4a, and results in a non-canonical senescence-associated secretory phenotype, which is pro-fibrotic and pro-hypertrophic. Pharmacological or genetic clearance of senescent cells in mice alleviates detrimental features of cardiac ageing, including myocardial hypertrophy and fibrosis. Our data describe a mechanism by which senescence can occur and contribute to age-related myocardial dysfunction and in the wider setting to ageing in post-mitotic tissues.

Mitochondrion-processed TERC regulates senescence without affecting telomerase activities.

Mitochondrial dysfunctions play major roles in ageing. How mitochondrial stresses invoke downstream responses and how specificity of the signaling is achieved, however, remains unclear. We have previously discovered that the RNA component of Telomerase TERC is imported into mitochondria, processed to a shorter form TERC-53, and then exported back to the cytosol. Cytosolic TERC-53 levels respond to mitochondrial functions, but have no direct effect on these functions, suggesting that cytosolic TERC-53 functions downstream of mitochondria as a signal of mitochondrial functions. Here, we show that cytosolic TERC-53 plays a regulatory role on cellular senescence and is involved in cognition decline in 10 months old mice, independent of its telomerase function. Manipulation of cytosolic TERC-53 levels affects cellular senescence and cognition decline in 10 months old mouse hippocampi without affecting telomerase activity, and most importantly, affects cellular senescence in terc-/- cells. These findings uncover a senescence-related regulatory pathway with a non-coding RNA as the signal in mammals.