FIG 2 - uploaded by Michel M Ouellette
Content may be subject to copyright.
Telomerase activity and TRF length decline over time. Telomerase activity, expressed relative to the reference adenocarcinoma cell line H1299, progressively declined during the first 100 doublings following transfection and then stabilized at a small percentage of H1299 levels. TRF length also declined before stabilizing at approximately 4 kb in most clones. Multiple measurements of the same sample were often made, indicating the variability of both the PCR-based TRAP assay and the analysis of telomere sizes on agarose gels.
Source publication
Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250-400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells con...
Contexts in source publication
Context 1
... activity was followed at multiple time points using the PCR-based TRAP assay (11). Activity fell progres- sively over the first 100 doublings following transfection, and then stabilized at relatively low levels (Fig. 2). This decrease in expression is expected for plasmid based expression systems which are thought to become methylated over time (31). On average, activity decreased from 20 -80% of the activity pres- ent in the control lung adenocarcinoma reference cell line H1299 to 1-5%. The progressive decrease in telomerase levels was accompanied by ...
Context 2
... over time (31). On average, activity decreased from 20 -80% of the activity pres- ent in the control lung adenocarcinoma reference cell line H1299 to 1-5%. The progressive decrease in telomerase levels was accompanied by decreased telomere lengths. Fig. 3 shows TRF sizes for clone B34 between population doubling levels 82 and 302, while Fig. 2 summarizes the data for all of the four ...
Similar publications
Stable transfection manipulation with antibiotic selection and passaging induces progressive cellular senescence phenotypes. However, the underlying mechanisms remain poorly understood. This study demonstrated that stable transfection of the empty vector induced PANC-1 cells into cellular senescence. Metabolomics revealed several acylcarnitines and...
We previously reported that TAK1, one of the mitogen-activated protein kinase kinase kinases (MAP3Ks), represses the transcription
of the human telomerase reverse transcriptase (hTERT) gene in human cancer cells and induces cellular senescence in normal diploid human cells. On the basis of these results,
we presumed a link between hTERT repression...
There is a striking interaction of genes and environment in the etiology of type 2 diabetes mellitus (T2DM). While endocrine disrupting chemicals (EDCs) like bisphenol-A (BPA) have received special attention for their mechanistic role in metabolic disruption, there is a lack of clinically relevant data on BPA levels in Asian Indians, a population w...
Aging is the major risk factor for cancer, cardiovascular disease, diabetes, and neurodegenerative disorders. Although we are far from understanding the biological basis of aging, research suggests that targeting the aging process itself could ameliorate many age-related pathologies. Senescence is a cellular response characterized by a stable growt...
The role of telomere shortening in the induction of replicative cellular senescence (CS) is well known and as a result, the involvement of telomerase and in particular its catalytic subunit, the telomerase reverse transcriptase (TERT) in CS has also been investigated. However, the majority of studies were conducted on cells that generally express h...
Citations
... It is known that cancer cells are characterized by high telomerase activity and tumor malignancy correlates with the level of telomerase activity [130,131]; the level of activity can vary hundredfold [132]. It could be hypothesized that if telomerase activity is relatively low or erratic, it will only 'heal' dysfunctional, critically shortened telomeres, thereby reducing DDR and SASP and reducing inflammation, but not enabling long-term growth [133]. The protective effect of telomerase under oxidative stress is also likely to be beneficial [61,[134][135]. ...
The science of telomeres and telomerase has made tremendous progress in recent decades. In this review, we consider it first in a historical context (the Carrel–Hayflick–Olovnikov–Blackburn chain of discoveries) and then review current knowledge on the telomere structure and dynamics in norm and pathology. Central to the review are consequences of the telomere shortening, including telomere position effects, DNA damage signaling, and increased genetic instability. Cell senescence and role of telomere length in its development are discussed separately. Therapeutic aspects and risks of telomere lengthening methods including use of telomerase and other approaches are also discussed.
... The signal intensity could reflect the number of repeats in the certain molecular weight. Average TRF length could be obtained by weighted calculation [27]. ...
Background
The mitochondrial gene MCCC2, a subunit of the heterodimer of 3-methylcrotonyl-CoA carboxylase, plays a pivotal role in catabolism of leucine and isovaleric acid. The molecular mechanisms and prognostic value still need to be explored in the context of specific cancers, including colorectal cancer (CRC).
Methods
In vitro and in vivo cell-based assays were performed to explore the role of MCCC2 in CRC cell proliferation, invasion, and migration. Mitochondrial morphology, membrane potential, intracellular reactive oxygen species (ROS), telomerase activity, and telomere length were examined and analyzed accordingly. Protein complex formation was detected by co-immunoprecipitation (CO-IP). Mitochondrial morphology was observed by transmission electron microscopy (TEM). The Cancer Genome Atlas (TCGA) CRC cohort analysis, qRT-PCR, and immunohistochemistry (IHC) were used to examine the MCCC2 expression level. The association between MCCC2 expression and various clinical characteristics was analyzed by chi-square tests. CRC patients’ overall survival (OS) was analyzed by Kaplan–Meier analysis.
Results
Ectopic overexpression of MCCC2 promoted cell proliferation, invasion, and migration, while MCCC2 knockdown (KD) or knockout (KO) inhibited cell proliferation, invasion, and migration. MCCC2 KD or KO resulted in reduced mitochondria numbers, but did not affect the gross ATP production in the cells. Mitochondrial fusion markers MFN1, MFN2, and OPA1 were all upregulated in MCCC2 KD or KO cells, which is in line with a phenomenon of more prominent mitochondrial fusion. Interestingly, telomere lengths of MCCC2 KD or KO cells were reduced more than control cells. Furthermore, we found that MCCC2 could specifically form a complex with telomere binding protein TRF2, and MCCC2 KD or KO did not affect the expression or activity of telomerase reverse transcriptase (TERT). Finally, MCCC2 expression was heightened in CRC, and patients with higher MCCC2 expression had favorable prognosis.
Conclusions
Together, we identified MCCC2 as a novel mediator between mitochondria and telomeres, and provided an additional biomarker for CRC stratification.
... In gel hybridization analysis of the DNA samples was performed as follows [54]: The gel was dried for more than 1h at room temperature. For native in-gel hybridization, gels were hybridized in hybridization buffer (10 × Denhard's buffer, 2μg/mL sonicated Escherichia coli DNA, 0.5% SDS and 5 × SSC) with 32 P-labled C-/G-rich telomeric probe. ...
Author summary
Telomeres are repetitive DNA: protein structures that protect linear chromosome ends from being recognized as double-strand DNA breaks. Telomeres bear very high loads of replication stress, and are essential for chromosomal stability and genome stability. Telomeric repeat-containing RNA (TERRA) is transcribed from subtelomeres to telomeres, and is prone to form R-loop structures by invading into telomeric DNA. In telomerase-negative human cancer cell lines, which maintain telomere length by homologous recombination-based alternative lengthening of telomeres (ALT) mechanism, both TERRA levels and telomeric R-loops are upregulated. Excessive telomere R-loops result in telomere instability. Therefore, only limited number of TERRA is required for telomere homeostasis. Here we show that, the RNA binding motif protein encoded on the X chromosome (RBMX), also known as hnRNP G, is a novel regulator of TERRA level and telomere integrity. Our data point to a completely new role of RBMX in TERRA expression regulation and telomere integrity maintenance, and open up a potential way for cancer therapy. In addition, this study also raises the possibility that RBMX also targeted other nuclear RNA to nuclear exosome for degradation.
... A common model for telomere length regulation (Fig. 4) poses that long telomeres contain more TRF1/TIN2/ POT1, so that within each nucleus, telomerase is blocked at long telomeres but not at the short ones (for review, see Hockemeyer and Collins 2015). Indeed, mammalian telomerase extends the shortest telomeres preferentially (Zhu et al. 1998;Ouellette et al. 2000). Chromatin immunoprecipitation (ChIP) experiments showed that the density of these three proteins per kilobase of telomeric DNA is the same in cells with long and short telomeres (Loayza and de Lange 2003). ...
It has been known for decades that telomerase extends the 3′ end of linear eukaryotic chromosomes and dictates the telomeric repeat sequence based on the template in its RNA. However, telomerase does not mitigate sequence loss at the 5′ ends of chromosomes, which results from lagging strand DNA synthesis and nucleolytic processing. Therefore, a second enzyme is needed to keep telomeres intact: DNA polymerase α/Primase bound to Ctc1–Stn1–Ten1 (CST). CST–Polα/Primase maintains telomeres through a fill-in reaction that replenishes the lost sequences at the 5′ ends. CST not only serves to maintain telomeres but also determines their length by keeping telomerase from overelongating telomeres. Here we discuss recent data on the evolution, structure, function, and recruitment of mammalian CST–Polα/Primase, highlighting the role of this complex and telomere length control in human disease.
... Cell and animal models with genetically modified or no telomerase activity have been especially helpful in uncovering the importance of telomerase in telomere maintenance. Population doubling in human fibroblasts causes telomere shortening in vitro (Harley et al., 1990), yet exogenous Tert treatment considerably extends their lifespan by maintaining critically short telomeres (Bodnar et al., 1998;Ouellette et al., 2000). Further, increased TERT expression preferentially protects short, dysfunctional telomeres to buffer senescence and avert malignant transformation (Sun et al., 2019). ...
Telomerase preserves genomic integrity by maintaining and protecting the telomeres. Seminal findings from 1985 revealed the canonical role of telomerase and motivated investigations into potential therapeutic strategies to combat one of the hallmarks of ageing—telomere attrition. Since then, the field of telomere biology has rapidly expanded, with telomerase serving essential roles in cancer and cell development through its canonical function. However, telomerase also exerts critical extra-telomeric functions through its protein (telomerase reverse transcriptase, TERT) and RNA components (telomerase RNA component, TERC). Telomerase re-activation or ectopic expression promotes survival and permits unlimited proliferation in tumours and healthy non-malignant cells. TERT gene therapies improve health and lifespan in ageing mice and mouse models of age-related diseases. The extra-telomeric functions of telomerase are critical to ageing. These include protection against oxidative stress, orchestration of chromatin modifications and transcription, and regulation of angiogenesis and metabolism (e.g. mitochondrial function and glucose control). Given these biological functions are key adaptations to endurance training and the recent meta-analytical findings that indicate exercise up-regulates TERT and telomerase, a comprehensive discussion on the implications of the canonical and extra-telomeric roles of telomerase is warranted. This review highlights the therapeutic benefits of telomerase-based treatments for idiopathic and chronic diseases that are linked to ageing. Discussion on the canonical and extra-telomeric roles of telomerase are presented, followed by a detailed summary of the evidence on how exercise influences telomerase. Finally, the potential cell signalling underpinning the exercise-induced modulation of telomerase are discussed with directions for future research.
... They assumed that increasing telomerase activity can lead to telomere lengthening by influencing telomeres, telomerase function, and senescence. The hypothesis was that modest and potentially intermittent telomerase activity would allow cells to only repair telomeres, lowering the DNA damage response and SASP, while decreasing inflammation and oxidative stress levels [96]. ...
... Gene therapy using adeno-associated viruses to introduce the telomerase gene into aging mice appears to be promising, with incremental improvements in several biomarkers [96][97][98][99]. More particularly, beneficial effects were observed on insulin sensitivity, osteoporosis, neuromuscular coordination, and several molecular biomarkers of aging with a significant increase in median lifespan [98]. ...
Coronary artery disease (CAD) is a multifactorial disease with a high prevalence, particularly in developing countries. Currently, the investigation of telomeres as a potential tool for the early detection of the atherosclerotic disease seems to be a promising method. Telomeres are repetitive DNA sequences located at the extremities of chromosomes that maintain genetic stability. Telomere length (TL) has been associated with several human disorders and diseases while its attrition rate varies significantly in the population. The rate of TL shortening ranges between 20 and 50 bp and is affected by factors such as the end-replication phenomenon, oxidative stress, and other DNA-damaging agents. In this review, we delve not only into the pathophysiology of TL shortening but also into its association with cardiovascular disease and the progression of atherosclerosis. We also provide current and future treatment options based on TL and telomerase function, trying to highlight the importance of these cutting-edge developments and their clinical relevance.
... This finding was unexpected and as far as we know, no similar changes in the time between divisions have been associated with immortalization. However, the transduction efficiencies of T-antigen-encoding vectors may be low and its expression may be transient, and therefore, transformation associated with inconsistent delivery and/or expression could produce genomic variability and morphological changes (Mayne et al., 1986;Ouellette et al., 2000), which could explain the increase in time between divisions of immortalized cells. ...
Müller cells are the principal glial cells in the retina and they assume many of the functions carried out by astrocytes, oligodendrocytes and ependymal cells in other regions of the central nervous system. Müller cells express growth factors, neurotransmitter transporters and antioxidant agents that could fulfill important roles in preventing excitotoxic damage to retinal neurons. Vertebrate Müller cells are well-defined cells, characterized by a common set of features throughout the phylum. Nevertheless, several major differences have been observed among the Müller cells in distinct vertebrates, such as neurogenesis, the capacity to reprogram fish Müller glia to neurons. Here, the Müller glia of the largest adult mammal in the world, the whale, have been analyzed, and given the difficulties in obtaining cetacean cells for study, these whale glia were analyzed both in primary cultures and as immortalized whale Müller cells. After isolating the retina from the eye of a beached sei whale (Balaenoptera borealis), primary Müller cell cultures were established and once the cultures reached confluence, half of the cultures were immortalized with the simian virus 40 (SV40) large T-antigen commonly used to immortalize human cell lines. The primary cell cultures were grown until cells reached senescence. Expression of the principal molecular markers of Müller cells (GFAP, Vimentin and Glutamine synthetase) was studied in both primary and immortalized cells at each culture passage. Proliferation kinetics of the cells were analyzed by time-lapse microscopy: the time between divisions, the time that cells take to divide, and the proportion of dividing cells in the same field. The karyotypes of the primary and immortalized whale Müller cells were also characterized. Our results shown that W21M proliferate more rapidly and they have a stable karyotype. W21M cells display a heterogeneous cell morphology, less motility and a distinctive expression of some typical molecular markers of Müller cells, with an increase in dedifferentiation markers like α-SMA and β-III tubulin, while they preserve their GS expression depending on the culture passage. Here we also discuss the possible influence of the animal’s age and size on these cells, and on their senescence.
... The canonical function of TERT is the synthesis of telomeric repeats and the maintenance of telomere integrity. TERT is expressed at higher levels than needed for maintaining telomere length, indicating it has other adaptive functions (Ouellette et al. 2000). To date, accumulating studies suggest that TERT plays fundamental functions in regulation of DNA damage responses, promotion of cell growth, and regulation of cell metabolism, independent of its canonical functions (Romaniuk et al. 2018; Thompson and Wong 2020;Zhou et al. 2014). ...
The telomerase reverse transcriptase (TERT) is the core catalytic subunit of telomerase. Its canonical function is synthesizing telomeric repeats to maintain telomere length and chromosomal stability. Accumulating evidence suggests that TERT has other important fundamental functions in addition to its catalytic telomere repeat synthesis activity. However, the non-canonical roles of TERT independent of its enzymatic activity are not clear in filamentous fungi. In the present study, we characterized the GlTert gene in Ganoderma lucidum. The non-canonical roles of GlTert were explored using GlTert-silenced strains (Terti8 and Terti25) obtained by RNA interference. Silencing GlTert delayed the fungal growth, decreased the length between hyphal branches, and induced fungal resistance to oxidative stress in G. ludicum. Further examination revealed that the intracellular ROS (reactive oxygen species) levels were increased while the enzyme activities of the antioxidant systems (superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase) were decreased in GlTert-silenced strains. In addition, silencing GlTert decreased the ganoderic acid (GA) biosynthesis of G. lucidum. Taken together, our results indicate that GlTert plays a fundamental function on fungal growth, oxidative stress, and GA biosynthesis in G. lucidum, providing new insights for the canonical functions of TERT in filamentous fungi.
Key points
• GlTert affected fungal growth and hyphal branching of G. lucidum.
• Silencing GlTert increased the intracellular ROS levels of G. lucidum.
• GlTert regulated GA biosynthesis ofG. lucidum.
... Last, there are inherent limitations associated with the MMqPCR method (42) employed in the current study. While the qPCR platform enables a highly sensitive and quantitative assessment of telomere "length," it does not allow for direct length measurement as in the TRF method (73,74). Instead, the MMqPCR measures the "amount" of telomere repeats present. ...
Context
Chronic exposure to glucocorticoids (GCs) or stress increases the risk of medical disorders, including cardiovascular and neuropsychiatric disorders. GCs contribute to an accelerated aging, while the link between chronic GCs exposure and disease onset is well established, the underpinning mechanisms are not clear.
Objective
we explored the potential nexus between GCs or stress exposure and telomere length.
Design, Setting, and Participants
rats exposed to three weeks of chronic stress; an iatrogenic mouse model of Cushing’s syndrome (CS); a mouse neuronal cell line; 33 patients with CS and 75 healthy human people were studied.
Results
1.Telomere length is associated with exposure to stress in rats: 54.5% (P=0.036) reduction in telomere length in the stressed animals. Genomic DNA extracted from the dentate gyrus of stressed and unstressed rats showed 43.2% reduction in telomere length (P=0.006). 2. Mice were exposed to corticosterone (CORT), this treatment produced a 61.4% reduction in telomere length in the blood gDNA (P=5.75x 10-5). 3. We observed a 40.8% reduction in the telomere length in patients with active CS compared to healthy controls (P=0.006). There was a 17.8% reduction in telomere length in cured CS patients, no different from controls (P=0.08). For both cured and active CS, telomere length correlated significantly with the duration of hypercortisolism (R2=0.22, P=0.007). 4. There was a 27.6% reduction in telomere length between low vs. high tertiles in bedtime cortisol levels (P=0.019).
Conclusions
Our findings demonstrate that exposure to stress and/or glucocorticoids is associated with shortened telomeres, and that shortening may be partially reversible.
... The efficacy of anti-telomerase therapy depends on the initial shortest telomere length which dictates the onset of telomere dysfunction [30]. As tumor cells exhibit short telomere and very little telomerase could maintain the shortest telomeres [31], highly potent inhibitors are needed. In this study, we found that high DKC1 expression is associated with LUAD progression and indicates poor prognosis. ...
Background
Lung cancer is one of the most widely spread cancers in the world and half of the non-small cell lung cancers are lung adenocarcinoma (LUAD). Although there were several drugs been approved for LUAD therapy, a large portion of LUAD still cannot be effectively treated due to lack of available therapeutic targets. Here, we investigated the oncogenic roles of DKC1 in LUAD and its potential mechanism and explored the possibility of targeting DKC1 for LUAD therapy.
Methods
The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas Program (TCGA) databases were used to examine the DKC1 transcript levels. Gene expression with clinical information from tissue microarray of LUAD were analyzed for associations between DKC1 expression and LUAD prognosis. In addition, loss- and gain-of-function assays were used for oncogenic function of DKC1 both in vitro and in vivo.
Results
DKC1 is overexpressed in LUAD compared with adjacent normal tissues. High expression of DKC1 predicts the poor overall survival. DKC1 knockdown in LUAD cell lines induced G1 phase arrest and inhibited cell proliferation. Ectopic expression of DKC1 could rescue the growth of LUAD cell lines. In addition, the abundance of DKC1 is positively correlated with telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) levels in LUAD. DKC1 downregulation resulted in decreased TERC expression, reduced telomerase activity and shorten telomere, and thus eventually led to cell senescence and apoptosis.
Conclusions
Our results show that high DKC1 expression indicates poor prognosis of LUAD and DKC1 downregulation could induce telomere-related cell senescence and apoptosis. This study suggests that DKC1 could serve as a candidate diagnostic biomarker and therapeutic target for LUAD.
