Telomeric Repeat Amplification Protocol Research Articles

TERT Expression and Clinical Outcome in Pulmonary Carcinoids.

The clinical course of pulmonary carcinoids ranges from indolent to fatal disease, suggesting that specific molecular alterations drive progression toward the fully malignant state. A similar spectrum of clinical phenotypes occurs in pediatric neuroblastoma, in which activation of telomerase reverse transcriptase (TERT) is decisive in determining the course of disease. We therefore investigated whether TERT expression defines the clinical fate of patients with pulmonary carcinoid. TERT expression was examined by RNA sequencing in a test cohort and a validation cohort of pulmonary carcinoids (n = 88 and n = 105, respectively). A natural TERT expression cutoff was determined in the test cohort on the basis of the distribution of TERT expression, and its prognostic value was assessed by Kaplan-Meier survival estimates and multivariable analyses. Telomerase activity was validated by telomere repeat amplification protocol assay. Similar to neuroblastoma, TERT expression exhibited a bimodal distribution in pulmonary carcinoids, separating tumors into TERT-high and TERT-low subgroups. A natural TERT cutoff discriminated unfavorable from favorable clinical courses with high accuracy both in the test cohort (5-year overall survival [OS], 0.547 ± 0.132 v 1.0; P < .001) and the validation cohort (5-year OS, 0.788 ± 0.063 v 0.913 ± 0.048; P < .001). In line with these findings, telomerase activity was largely absent in TERT-low tumors, whereas it was readily detectable in TERT-high carcinoids. In multivariable analysis considering TERT expression, histology (typical v atypical carcinoid), and stage (≤IIA v ≥IIB), high TERT expression was an independent prognostic marker for poor survival, with a hazard ratio of 5.243 (95% CI, 1.943 to 14.148; P = .001). Our data demonstrate that high TERT expression defines clinically aggressive pulmonary carcinoids with fatal outcome, similar to neuroblastoma, indicating that activation of TERT may be a defining feature of lethal cancers.

Preparation and anticancer activity of telomerase inhibitor TAT-LPTS39 polypeptide.

Telomerase is activated in most cancer cells, and thus telomerase is an ideal target for cancer therapy. The human liver-associated candidate tumour suppressor LPTS/PinX1, is the only human protein reported to bind with the telomerase catalytic subunit telomerase reverse transcriptase (TERT) and inhibit telomerase activity. The C-terminal fragment of LPTS/PinX1 (LPTS/PinX1290-328) contains a telomerase inhibitory domain that is needed for inhibition of telomere elongation and induction of apoptosis. This study prepared the TAT-LPTS39 (TAT-LPTS/PinX1290-328) polypeptide and analysed its effect of the tumour growth. LPTS/PinX1290-328 was fused with TAT [11 amino acid (aa) peptide of the HIV transactivator of transcription protein] to generate the recombinant protein GST-TAT-LPTS39 and was transduced into cells. Telomerase activity was identified by the telomeric repeat amplification protocol (TRAP) and the relative telomere length (RTL) was measured by quantitative real-time polymerase chain reaction (qPCR). The effects of the TAT-LPTS39 protein on cell growth and death were evaluated by 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT), cell culture doubling time and flow cytometry assays. The cell derived xenograft (CDX) model was used to examine tumour growth inhibition effect of TAT-LPTS39 polypeptide in vivo. We successfully expressed and purified the recombinant protein GST-TAT-LPTS39 in vitro. The GST-TAT-LPTS39 protein was efficiently delivered into cells, inhibited telomerase activity and the growth of the telomerase-positive liver cancer cells BEL-7404 and QGY7701, and induced the senescence and apoptosis in telomerase-positive Hela, BEL-7404 and QGY7701 cells, but was ineffective to telomerase-negative cells in vitro. The TAT-LPTS39 polypeptide without the GST tag similarly inhibited the growth of telomerase-positive cancer cells Hela and PLC-PRF-5 in vitro, BEL-7404 CDX tumour in vivo and shortened telomere length. The TAT-LPTS39 polypeptide has the ability to inhibit telomerase activity and suppress the growth of all tested human telomerase-positive cancer cells in vitro and in vivo, suggesting a potential anticancer drug development.

All-trans-retinoic acid modulates glycolysis via H19 and telomerase: the role of mir-let-7a in estrogen receptor-positive breast cancer cells

BackgroundBreast cancer (BC) is the most commonly diagnosed cancer in women. Treatment approaches that differ between estrogen-positive (ER+) and triple-negative BC cells (TNBCs) and may subsequently affect cancer biomarkers, such as H19 and telomerase, are an emanating delight in BC research. For instance, all-trans-Retinoic acid (ATRA) could represent a potent regulator of these oncogenes, regulating microRNAs, mostly let-7a microRNA (miR-let-7a), which targets the glycolysis pathway, mainly pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA) enzymes. Here, we investigated the potential role of ATRA in H19, telomerase, miR-let-7a, and glycolytic enzymes modulation in ER + and TNBC cells.MethodsMCF-7 and MDA-MB-231 cells were treated with 5 µM ATRA and/or 100 nM fulvestrant. Then, ATRA-treated or control MCF-7 cells were transfected with either H19 or hTERT siRNA. Afterward, ATRA-treated or untreated MDA-MB-231 cells were transfected with estrogen receptor alpha ER(α) or beta ER(β) expression plasmids. RNA expression was evaluated by RT‒qPCR, and proteins were assessed by Western blot. PKM2 activity was measured using an NADH/LDH coupled enzymatic assay, and telomerase activity was evaluated with a quantitative telomeric repeat amplification protocol assay. Student’s t-test or one-way ANOVA was used to analyze data from replicates.ResultsOur results showed that MCF-7 cells were more responsive to ATRA than MDA-MB-231 cells. In MCF-7 cells, ATRA and/or fulvestrant decreased ER(α), H19, telomerase, PKM2, and LDHA, whereas ER(β) and miR-let-7a increased. H19 or hTERT knockdown with or without ATRA treatment showed similar results to those obtained after ATRA treatment, and a potential interconnection between H19 and hTERT was found. However, in MDA-MB-231 cells, RNA expression of the aforementioned genes was modulated after ATRA and/or fulvestrant, with no significant effect on protein and activity levels. Overexpression of ER(α) or ER(β) in MDA-MB-231 cells induced telomerase activity, PKM2 and LDHA expression, in which ATRA treatment combined with plasmid transfection decreased glycolytic enzyme expression.ConclusionsTo the best of our knowledge, our study is the first to elucidate a new potential interaction between the estrogen receptor and glycolytic enzymes in ER + BC cells through miR-let-7a.

Functional Testing of Variants of Uncertain Significance in TERC, TERT,and RTEL1 from Adult Patients with Telomere Biology Disorders

INTRODUCTION Telomere biology disorders (TBDs) are multisystem diseases caused by accelerated telomere shortening that can present with bone marrow failure and pulmonary fibrosis, among other abnormalities. TBD diagnosis is considered when average blood telomere length (TL) measured by clinical flowFISH is &amp;lt;1 st percentile and/or a pathogenic variant in telomere maintenance genes (most commonly TERT, TERC, RTEL1) is identified. Pathogenic variants in genes coding for the telomerase protein ( TERT) and RNA component ( TERC) decrease telomerase activity. Similarly, variants in RTEL1 result in defective unwinding of the t-loop at telomere ends, increasing the levels of free t-circles in cells. Variants in these genes are often classified as variants of uncertain significance (VUSs by ACMG criteria) with additional efforts needed for variant curation. To address this, we describe results of functional testing performed in TBD patients with VUSs involving TERT, TERC, and RTEL1. METHODS TBD suspected patients were identified through the Pre-Myeloid Cancer and Bone Marrow Failure Clinic (Mayo Clinic) where they underwent clinical flowFISH and genetic testing for TBD related genes. Informed consent was obtained with approval from the Mayo Clinic Institutional Review Board. Viable peripheral blood and bone marrow samples were collected along with age matched healthy volunteers. Telomerase activity was measured in samples with TERT and TERC variants through telomeric repeat amplification protocol (TRAP) assays using the TRAPeze XL Telomerase Detection Kit (MilliporeSigma) following manufacturer instructions. HCT116 cells were used as positive controls while heat inactivated samples served as negative controls. Fluorescence was measured in a SpectraMax plate reader. Percentage of total product generated (TPG) relative to healthy age matched controls was calculated. T-circle detection assays were used on RTEL1 samples based on the protocol by Zhang et al. (2017). Final southern blotting was completed using the TeloTAGGG Telomere Length Assay Kit (Roche), and t-circle bands were visualized in a c600 GEL Imaging System (Azure Biosystems). RESULTS Ten patients (mean age = 53 years (27-71), 50% female) were included in our study. Patients 1 to 5 presented VUS in TERT (n=4) or TERC (n=1) and TL &amp;lt;1 st percentile in lymphocytes and/or granulocytes. Patient 6 carried a known pathogenic variant in RTEL1 and was included as a positive control. Patients 7 to 9 with RTEL1 variants presented TL close to the 10 th or between the 1 st-10 th percentile. No TL results were available for patient 10 but was tested due to the presence of a VUS in RTEL1 (Table 1). Similarly, while patient 7 carried a likely pathogenic variant, it was tested due to presenting lymphocyte TL between the 1 st-10 th percentile. TRAP results indicated decreased telomerase activity compared to age matched controls in all samples with TERT and TERC variants suggesting a pathogenic effect (52.%, 10.7%, 48.7%, 4.4% and 5.2% of TPG compared to age matched controls, respectively) (Table 1). On the other hand, t-circles were only seen in patient 6 (Figure 2). DISCUSSION We used TRAP and t-circle assays to explore the telomerase activity and presence of t-circles in patient samples with TERT/TERC and RTEL1 variants. In the case of TERT/TERC, we observed a reduction of telomerase activity in all samples. Together with blood TL below the 1 st percentile, this suggests that these variants are indeed deleterious (Table 1). However, since patient 4 presented two TERT VUSs, additional testing is needed to conclude how each variant impacts telomerase activity independently. In regard to RTEL1 variants, we observed t-circles in patient 6 as expected. No t-circles could be seen on all remaining samples, including patient 7 who carried a variant classified as likely pathogenic (Figure 1). This in-frame deletion has been described once in a TBD family (Cogan et al. 2015) but was not functionally tested. This result, together with the borderline TL seen in this patient, may suggest that some compensatory mechanism exists on this sample, or that the deleterious effect of the variant is not strong enough to be detected by our assay. Further studies are currently ongoing by our group to validate these results using a cell-based transfection disease model. In summary, we show that functional testing can be a helpful tool to determine pathogenicity of VUSs in TERT/TERC and RTEL1.

A Zebrafish Model of Parn-Deficiency Reveals Conserved Mechanisms Underlying Marrow Failure and Opportunities for Therapeutic Amelioration of Hematopoiesis

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome (IBMFS) and a telomere biology disorder that is characterized by physical malformations such as nail dystrophy, oral leukoplakia, reticular hyperpigmentation, multilineage cytopenia and immunodeficiency. Definitive treatment for the hematological complications is hematopoietic stem cell transplantation (HSCT), which is associated with high risk of organ toxicity and engraftment failure in this population. Further, DC patients are prone to developing myelodysplastic syndrome (MDS) and solid tumors. Germline mutations in the poly(A)-specific ribonuclease (PARN) gene are associated with Hoyeraal-Hreidarsson syndrome, the most severe form of DC. PARN encodes a deadenylase enzyme involved in the 3'-end processing of various RNAs including the telomerase RNA component (TERC). The clinical heterogeneity of phenotypes and the paucity of primary human samples necessitate the use of animal models such as zebrafish ( Danio rerio), given their highly conserved genetic program, to elucidate underlying biology. Zebrafish have a single PARN ortholog with 64% sequence identity at the amino acid level. Using CRISPR-Cas9 mutagenesis, we generated a 1.3 kb deletion in the zebrafish parn gene, predicted to result in premature protein truncation. To measure the hematopoietic output of the parn mutants, we performed whole-mount in situ hybridization (WISH) for key hematopoietic lineage markers. At 48 hours post-fertilization (hpf), we found a reduction in lcp1 + total leukocytes, mpx + neutrophils and hbbe3 + mature erythrocytes. Impaired erythroid differentiation was accompanied by an accumulation of gata1 + erythroid progenitors. This finding was further corroborated by o-dianisidine staining, showing reduced hemoglobinized erythrocytes. Thus, erythrocyte and leukocyte differentiation are compromised in our zebrafish parn mutant, phenocopying the multilineage cytopenia observed in DC patients. Flow cytometry of the whole kidney marrow (WKM, human bone marrow equivalent) at 12 months of age revealed an increase in hematopoietic stem and progenitor cells in the parn mutants. Giemsa staining of WKM touchpreps further identified an increase in myeloid-biased progenitors. Next, we investigated the consequences of Parn loss on telomerase activity. Using the telomere repeat amplification protocol, we found reduced telomerase activity in the WKMs of parn mutant zebrafish. Using 3' rapid amplification of cDNA ends, we observed longer ends in the terc amplicons of the parn mutants, reminiscent of defective deadenylation of human TERC RNA in the setting of PARN mutations. Exogenous thymidine supplementation has been shown to support telomere elongation in human cells. Following treatment of zebrafish embryos with thymidine nucleoside, we observed an improvement in erythrocyte numbers in the parn mutants by o-dianisidine staining at 48 hpf. Collectively, our preliminary findings suggest that telomerase-dependent mechanisms may contribute to the erythropenia seen in parn-mutant zebrafish. In summary, we generated a robust zebrafish model carrying a germline parn mutation that phenocopies the molecular and hematologic features seen in DC patients. Our model holds promise as a new in vivo tool for investigating DC-related disease mechanisms and as a platform for preclinical therapeutic screening.

In Vitro Assessment of Gold Nanoparticles on Telomerase Activity and Telomere Length in Human Fibroblasts.

Telomerase activity coincides with lengthening of the ends of chromosomes known as telomeres. Telomere length is used as a marker for cellular aging. Telomeres shorten over time as cells divide, and certain bioactive compounds such as gold nanoparticles (AuNPs) may slow the shortening of telomeres by increasing telomerase activity. The objective of the present study is to assess the effect of AuNPs on telomerase activity and telomere length in human fibroblasts. Telomerase activity was measured using enzyme-linked immunosorbent assay (ELISA) in primary human lung fibroblasts (IMR90) and using quantitative PCR-based telomeric repeat amplification protocol (Q-TRAP) in primary human dermal fibroblasts, neonatal (HDFn). Telomere length was determined by Telomere Analysis Technology (TAT®)assay in HDFn. In IMR90, all AuNP treatments showed significant increases in telomerase activity when compared to earlier passages. HDFn treated with AuNPs at 0 ppm, 0.05 ppm, 0.5 ppm, or 5 ppm did not show significant differences in telomerase activity compared to the control group. Significant differences in telomere length in HDFn were observed at 2 weeks of 0.05 and 0.5 ppm AuNPs under oxidative culture conditions as compared to the control group. The study showed preliminary evidence that AuNPs may increase telomerase activity and decelerate the shortening of telomeres in human fibroblasts, suggesting its potential anti-aging effects, which warrants further investigation.

Digital CRISPR/Cas12a-based platform for precise quantification of telomerase activity

Precise quantification of telomerase activity is of great significance for telomerase-based early cancer diagnosis, as well as the screening of prodrugs. Telomeric repeat amplification protocol (TRAP) has been most widely used for assessing telomerase activity, which is limited by false-positive results, complicated operations, and long detection time. Herein, we develop a droplet digital hyperbranched amplification enhanced CRISPR/Cas12a-based telomerase detection platform (ddHECTelo) for precise quantitative detection of telomerase activity. By combining with the droplet digital platform and hyperbranched amplification, the ddHECTelo allows precise quantification of telomerase activity with single-molecule sensitivity. A polydisperse droplets digital system was employed, so that the reaction mixtures could be partitioned into picoliter-scale polydisperse droplets in several seconds to avoid the pre-amplification, without the need for a sophisticated microfluidic chip. This is the first digital isothermal platform for sensitive and precise quantification of telomerase activity, which shows great potential in telomerase-based early cancer diagnosis. It could also be used to analyze the telomerase activity in fundamental biomedical research.

Impaired telomere pathway and fertility in Senescence-Accelerated Mice Prone 8 females with reproductive senescence.

Ovarian aging is the main cause of infertility and telomere attrition is common to both aging and fertility disorders. Senescence-Accelerated Mouse Prone 8 (SAMP8) model has shortened lifespan and premature infertility, reflecting signs of reproductive senescence described in middle-aged women. Thus, our objective was to study SAMP8 female fertility and the telomere pathway at the point of reproductive senescence. The lifespan of SAMP8 and control mice was monitored. Telomere length (TL) was measured by in situ hybridization in blood and ovary. Telomerase activity (TA) was analyzed by telomere-repeat amplification protocol, and telomerase expression, by real-time quantitative PCR in ovaries from 7-month-old SAMP8 and controls. Ovarian follicles at different stages of maturation were evaluated by immunohistochemistry. Reproductive outcomes were analyzed after ovarian stimulation. Unpaired t-test or Mann-Whitney test were used to calculate p-values, depending on the variable distribution. Long-rank test was used to compare survival curves and Fisher's exact test was used in contingency tables. Median lifespan of SAMP8 females was reduced compared to SAMP8 males (p = 0.0138) and control females (p < 0.0001). In blood, 7-month-old SAMP8 females presented lower mean TL compared to age-matched controls (p = 0.041). Accordingly, the accumulation of short telomeres was higher in 7-month-old SAMP8 females (p = 0.0202). Ovarian TA was lower in 7-month-old SAMP8 females compared to controls. Similarly, telomerase expression was lower in the ovaries of 7-month-old SAMP8 females (p = 0.04). Globally, mean TL in ovaries and granulosa cells (GCs) were similar. However, the percentage of long telomeres in ovaries (p = 0.004) and GCs (p = 0.004) from 7-month-old SAMP8 females was lower compared to controls. In early-antral and antral follicles, mean TL of SAMP8 GCs was lower than in age-matched controls (p = 0.0156 for early-antral and p = 0.0037 for antral follicles). Middle-aged SAMP8 showed similar numbers of follicles than controls, although recovered oocytes after ovarian stimulation were lower (p = 0.0068). Fertilization rate in oocytes from SAMP8 was not impaired, but SAMP8 mice produced significantly more morphologically abnormal embryos than controls (27.03% in SAMP8 vs. 1.22% in controls; p < 0.001). Our findings suggest telomere dysfunction in SAMP8 females, at the time of reproductive senescence.

Lamin B1 regulates the growth of hepatocellular carcinoma cells by influencing telomerase activity

Lamin B1 (LMNB1) is highly expressed in liver cancer tissues, and its influence and mechanism on the proliferation of hepatocellular carcinoma cells were explored by knocking down the expression of the protein. In liver cancer cells, siRNAs were used to knock down LMNB1. Knockdown effects were detected by Western blotting. Changes in telomerase activity were detected by telomeric repeat amplification protocol assay (TRAP) experiments. Telomere length changes were detected by quantitative real-time polymerase chain reaction (qPCR). CCK8, cloning formation, transwell and wound healing were performed to detect changes in its growth, invasion and migration capabilities. The lentiviral system was used to construct HepG2 cells that steadily knocked down LMNB1. Then the changes of telomere length and telomerase activity were detected, and the cell aging status was detected by SA-β-gal senescence staining. The effects of tumorigenesis were detected by nude mouse subcutaneous tumorigenesis experiments, subsequent histification staining of tumors, SA-β-gal senescence staining, fluorescence in situ hybridization (FISH) for telomere analysis and other experiments. Finally, the method of biogenesis analysis was used to find the expression of LMNB1 in clinical liver cancer tissues, and its relationship with clinical stages and patient survival. Knockdown of LMNB1 in HepG2 and Hep3B cells significantly reduced telomerase activity, cell proliferation, migration and invasion abilities. Experiments in cells and tumor formation in nude mice had demonstrated that stable knockdown of LMNB1 reduced telomerase activity, shortened telomere length, senesced cells, reduced cell tumorigenicity and KI-67 expression. Bioinformatics analysis showed that LMNB1 was highly expressed in liver cancer tissues and correlated with tumor stage and patient survival. In conclusion, LMNB1 is overexpressed in liver cancer cells, and it is expected to become an indicator for evaluating the clinical prognosis of liver cancer patients and a target for precise treatment.

Cytotoxic and Anti-proliferative Effects of Moringa oleifera Lam. on HeLa Cells

Background:The aim of the study was to determine the mechanism of Moringa oleifera-induced apoptosis in HeLa cells. HeLa cells over-express cyclin E and cyclin B1, abrogate G0-G1 and G2-M cell cycle arrest, promoting tumorigenesis. Cyclin E, cyclin B1, E2F1 and telomerase expression, and caspase-3 and -7 activation were assessed after 24-treatment with M. oleifera leaf fractions.Material and methods:Apoptosis through caspase-3 and caspase-7 activation was determined quantitatively by the FAM FLICA™ Caspase-3/7 assay. Cyclin E, cyclin B1 and E2F1 were quantified by flow cytometry. Telomerase was evaluated by Telomeric repeat amplification protocol (TRAP reaction). The effects on colony formation were assessed by seeding treated cells in six-well plates for 7 days under culture conditions. The MTT assay was used to determine cell survival.Results:HeLa cells treated for 24 hours with M. oleifera leaf fractions showed dose-dependent cytotoxicity, activation of caspases-3 and -7, down regulation of cyclin E, cyclin B1, E2F1, and inhibition of telomerase expression. Cell cycle analysis of the dead cell population showed G2-M cellcycle arrest.Conclusion:M. oleifera leaf fractions triggered apoptosis through the mitochondrial pathway and cell cycle arrest at G2-M phase in HeLa cells after 24-hour treatment, through down regulation of cyclin E and cyclin B1 expression, and caspase-3 and -7 activation. In addition, M. oleifera leaf extract induces senescence in HeLa cells through the down-regulation of telomerase. Colony formation and cell proliferation were inhibited in a dose-dependent manner, corresponding with telomerase inhibition.

Telomere Length and Telomerase Activity in Subcutaneous and Visceral Adipose Tissues from Obese and Non-Obese Patients with and without Colorectal Cancer

To investigate the molecular mechanisms that link obesity and colorectal cancer (CRC), we analyzed parameters related to telomere function in subcutaneous and visceral adipose tissues (SAT and VAT), including subjects with and without CRC, who were classified according to their body mass index (BMI). Adipose tissues were obtained from 147 patients who had undergone surgery. A total of 66 cases corresponded to CRC patients, and 81 subjects were not affected by cancer. Relative telomere length was established by qPCR, and telomerase activity was determined by a method based on the telomeric repeat amplification protocol. Our results indicated longer telomeres in patients affected by CRC, both in SAT and VAT, when compared to the group of subjects without CRC. Tumor local invasion was associated with telomere length (TL) in SAT. Considering the BMI values, significant differences were found in the TL of both adipose tissues between subjects affected by CRC and those without cancer. Overweight subjects showed the greatest differences, with longer telomeres in the group of CRC patients, and a higher number of cases with telomerase reactivation in the VAT of subjects without cancer. In conclusion, parameters related to telomere function in adipose tissue could be considered as potential biomarkers in the evaluation of CRC and obesity.

Sensitive naked-eye detection of telomerase activity based on exponential amplification reaction and lateral flow assay.

Telomerase is a promising diagnostic and prognostic biomarker for cancers. Sensitive, simple, and reliable telomerase activity detection is vital for cancer diagnosis. Herein, we developed an ultrasensitive visualized assay for telomerase activity that combined the exponential amplification reaction (EXPAR) and lateral flow assay for easy and quick signal readout, which we termed as a lateral flow readout-EXPAR (LFR-EXPAR) assay. In the LFR-EXPAR assay, telomerase elongation products initiate the exponential amplification reaction, the generated trigger hybridizes with the reporter to form the recognition site of the nicking enzyme, and the nicking enzyme cuts the reporter strand. The degradation of the reporter can be detected with a universal lateral flow dipstick and read out with the naked eye. After conducting a series of proof-of-concept investigations, the LFR-EXPAR assay was found to achieve a sensitivity comparable to that of a TRAP (telomere repeat amplification protocol) assay. The LFR-EXPAR assay can be used to realize ultrasensitive and point-of-care detection of telomerase without requiring specialized instruments, holding great promise for early cancer diagnosis.

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

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

Monitoring Telomere Maintenance During Regeneration of Annelids.

Telomere shortening is a hallmark of aging and eventually constrains the proliferative capacity of cells. The protocols discussed here are used for monitoring telomeres comprehensively in Aeolosoma viride, a model system for regeneration studies. We present methods for analyzing the activity of telomerase enzyme in regenerating tissue by telomeric repeat amplification protocol (TRAP) assay, for comparing telomere length between existing tissue and newly regenerated tissue by telomere restriction fragment (TRF) assay, as well as for visualizing telomeres by fluorescence in situ hybridization (FISH).

Relationship between Protein kinase C isoforms, Telomerase and Alpha-fetoprotein through PI3K/AKT/mTOR pathway in Hepatocellular carcinoma

Protein kinase C (PKC) family has been an alluring objective for new cancer drug discovery. It has been reported to regulate telomerase in several cancer types. Our team had previously used telomerase to elucidate alpha-fetoprotein (AFP) modulation in hepatocellular carcinoma (HCC). The aim of this study was to investigate the interrelationships among PKC isoforms, telomerase and AFP in HCC. PKCα and PKCδ were the most expressed isoforms in HepG2/C3A, PLC/PRF/5, SNU-387 and SKOV-3 cells. Following the upregulation of AFP using pCMV3-AFP and the human telomerase reverse transcriptase (hTERT) using a construct expressing a wild-type hTERT, and after their inhibition with all-trans retinoic acid and hTERT siRNA each respectively, we found that the expression of PKCα, PKCβI, PKCβII and PKCδ was affected by the variation of AFP and hTERT mRNA levels. An increase in AFP expression and secretion was observed after gene silencing of PKCα, PKCβ, PKCδ, and PKCε in HepG2/C3A. A similar pattern was observed in transfected PLC/PRF/5 cells, however PKCδ isoform silencing decreased AFP expression. Furthermore, telomerase activity was quantified using quantitative telomeric repeat amplification protocol. The variations in hTERT expression and telomerase activity were similar to those of AFP. Further investigation showed that PKC isoforms regulate AFP and hTERT expression levels through PI3K/AKT/mTOR pathway in HepG2/C3A and PLC/PRF/5 cells. Thus, these results show for the first time a possible interrelationship that links PKC isoforms to both AFP and hTERT via PI3K/AKT/mTOR pathway in HCC.

Triazolyl Dibenzo[a,c]phenazines Stabilize Telomeric G-quadruplex and Inhibit Telomerase.

We herein report the synthesis and biophysical evaluation of triazolyl dibenzo[a,c]phenazine derivatives as a novel class of G-quadruplex ligands. The aromatic core facilitates π-π interaction and the flexible, protonatable side chains interact with the phosphate backbone of DNA via electrostatic interactions. Förster resonance energy transfer (FRET) melting assay and isothermal titration calorimetry (ITC) studies suggest that these ligands show binding preference for the hTELO G-quadruplex over G-quadruplexes found in the promoter region of various oncogenes and duplex DNA. The in vitro telomeric repeat amplification protocol (Q-TRAP) assay reveals that these ligands reduce telomerase activity in cancer cells.

Selective Apoptosis and Growth Impairment of Cancer Cells Induced by Human Telomerase Reverse Transcriptase (hTERT) Targeting Artificial MicroRNA

Human telomerase reverse transcriptase (hTERT) is a promising cancer target, and amiRNA particle displays the siRNA’s specificity and miRNA’s safety, suggesting that cancers can be treated more effective and safely by hTERT targeting amiRNA particles. Hela, NCI-H446, U2-OS and Huvec cells were transfected by hTERT targeting amiRNA particles. hTERT expression, telomerase activity and cell viability were evaluated by quantitative reverse transcription-PCR (qRT-PCR), western blot (WB), telomeric repeat amplification protocol (TRAP) assays, MTT method, transwell protocol, fluorescence-activated cell sorting (FACS) technologies, angiogenesis assay, and xenograft tumor models. Results: hTERT expression and telomerase activity in Hela and NCIH446 were significantly inhibited by amiRNA. Anti-proliferation and pro-apoptosis effects were only observed in transfected Hela and NCI-H446 cells, but anti-migration and anti-angiogenesis effects were presented in transfected Huvec cells. More interestingly, low to 1.56 nM amiRNA can inhibit the proliferation of Hela cells by 80.99±5.24%. Conclusion: amiRNA selectively and effectively impairs the growth, and assists the apoptosis of telomerase-positive cancer cells.

Characterization of tree shrew telomeres and telomerase

The use of tree shrews as experimental animals for biomedical research is a new practice. Several recent studies suggest that tree shrews are suitable for studying cancers, including breast cancer, glioblastoma, lung cancer, and hepatocellular carcinoma. However, the telomeres and the telomerase of tree shrews have not been studied to date. Here, we characterize telomeres and telomerase in tree shrews. The telomere length of tree shrews is approximately 23 kb, which is longer than that of primates and shorter than that of mice, and it is extended in breast tumor tissues according to Southern blot and flow-fluorescence in situ hybridization (FISH) analyses. Tree shrew spleen, bone marrow, testis, ovary, and uterus show high telomerase activities, which are increased in breast tumor tissues by telomeric repeat amplification protocol assays. The telomere length becomes shorter, and telomerase activity decreases with age. The tree shrew TERT and TERC are more highly similar to primates than to rodents. These findings lay a solid foundation for using tree shrews to study aging and cancers.

HTERT Downregulation Attenuates Resistance to DOX, Impairs FAK-Mediated Adhesion, and Leads to Autophagy Induction in Breast Cancer Cells.

Telomerase is known to contribute to telomere maintenance and to provide cancer cell immortality. However, numerous reports are showing that the function of the enzyme goes far beyond chromosome ends. The study aimed to explore how telomerase downregulation in MCF7 and MDA-MB-231 breast cancer cells affects their ability to survive. Consequently, sensitivity to drug resistance, proliferation, and adhesion were assessed. The lentiviral-mediated human telomerase reverse transcriptase (hTERT) downregulation efficiency was performed at gene expression and protein level using qPCR and Western blot, respectively. Telomerase activity was evaluated using the Telomeric Repeat Amplification Protocol (TRAP) assay. The study revealed that hTERT downregulation led to an increased sensitivity of breast cancer cells to doxorubicin which was demonstrated in MTT and clonogenic assays. During a long-term doubling time assessment, a decreased population doubling level was observed. Interestingly, it did not dramatically affect cell cycle distribution. hTERT downregulation was accompanied by an alteration in β1-integrin- and by focal adhesion kinase (FAK)-driven pathways together with the reduction of target proteins phosphorylation, i.e., paxillin and c-Src. Additionally, autophagy activation was observed in MDA-MB-231 cells manifested by alternations in Atg5, Beclin 1, LC3II/I ratio, and p62. These results provide new evidence supporting the possible therapeutic potential of telomerase downregulation leading to induction of autophagy and cancer cells elimination.

Detection of Urothelial Bladder Cancer Based on Urine and Tissue Telomerase Activity Measured by Novel RT-TRAP-2PCR Method.

Purpose: To assess the diagnostic performance of urine telomerase activity (TA) in detecting bladder cancer (BCa) using the modified Telomeric Repeat Amplification Protocol (TRAP) and the Real Time Telomeric Repeat Amplification Protocol with double Polymerase Chain Reaction (RT-TRAP-2PCR). Methods: In this case-control study, matching urine (in the pre- and post-surgical period) and tissue samples from 68 patients with BCa were assessed for TA. As a control, 45 urine samples were examined from non-BCa patients. TA levels were measured using TRAP and RT-TRAP-2 PCR methods. Results: Preoperative urinary TA was elevated in 64 (94.1%) of the 68 BCa patients. Urine TA was undetectable in 44 control patients, while TA was detected in one patient with histologically verified cystitis. Sensitivity for BCa detection of 94.1% and specificity of 97.8% were observed for urinary TA, while tissue TA had 100% sensitivity and 97.8% specificity. Both urine and tissue TA levels were not significantly higher in patients with muscle-invasive disease compared to those with non-muscle invasive BCa (p > 0.05). Urine and tissue TA levels were not associated with higher tumor grade, stage, and number of tumors (p > 0.05). However, the association was found between higher urinary and tissue TA levels with tumor size ≥ 3 cm (p = 0.02 and p = 0.01, respectively). During the first postoperative year, 17 BCa patients experienced disease recurrence, and urinary TA was present in 14 (82.4%) of these patients. The sensitivity and specificity of urinary TA levels for BCa recurrence in patients with non-muscle invasive bladder cancer (NMIBC) during follow-up were 82% and 94.4%, respectively. Conclusions: This pilot study demonstrates a high diagnostic performance of urinary and tissue TA levels measured by a new RT-TRAP-2PCR method for detecting and monitoring BCa. Additionally, the association was found between higher urinary and tissue TA levels with tumor size ≥ 3 cm; however, higher TA levels failed for significant correlation with advanced tumor stage and grade. Our study could serve as a benchmark for the evaluation of novel biomarkers using the RT-TRAP-2PCR method.