Telomeric Repeat Amplification Protocol Research Articles

Abatacept suppresses the telomerase activity of lymphocytes in patients with rheumatoid arthritis.

Telomere is a component of chromosomes that protects their ends from various stresses. The telomeres shorten during cell division, and their length is maintained by telomerase. The telomerase activity of lymphocytes was shown to be upregulated on lymphocyte activation, and abatacept was found to suppress the activation of T lymphocytes involved in pathogenesis of rheumatoid arthritis. Therefore, we investigated the effect of abatacept on lymphocyte telomerase activity in patients with rheumatoid arthritis. This study included 11 patients diagnosed with rheumatoid arthritis based on American College of Rheumatology 2010 criteria, who received abatacept treatment from August 2012 to August 2013. We collected their clinical data and obtained peripheral blood samples before starting abatacept, and 1, 3, 6, and 12months after treatment. Peripheral blood mononuclear cells were extracted using Ficoll density gradient centrifugation, and T and B lymphocytes were sorted by magnetic beads. The telomerase activity of lymphocytes was determined using the telomeric repeat amplification protocol. The telomerase activity of T lymphocytes declined from 0.357 to 0.161 (P<0.01) at 12months after abatacept treatment, and that of B lymphocytes declined from 0.554 to 0.202 (P<0.01). The telomerase activity of B lymphocytes, but not that of T lymphocytes, was also significantly downregulated 1month after treatment. Abatacept suppressed the telomerase activity of both T and B lymphocytes, although that of B lymphocytes was downregulated before T lymphocytes. These findings imply that the clinical efficacy of abatacept during the early phase depends on the suppression of B lymphocytes.

Telomerase stability and evaluation of real-time telomeric repeat amplification protocol

Telomerase is RNA directed polymerase which acts as reverse transcriptase based on its own RNA component. It is considered to be involved in the pathology of many diseases and is recognized as a potential biomarker. The aims were to determine the sample storage conditions and the time frame for samples analysis, then to prove reliability of enzyme activity measurement with real-time telomeric repeat amplification protocol (TRAP) and to evaluate the suitable standard samples for telomerase activity measurements. Samples used for stability and freeze-thaw study were peripheral blood leukocytes, obtained from apparently healthy persons, patients with diagnosed cancer and cell lines. Telomerase activity was measured using TRAP method, while standard evaluation was done using nuclear magnetic resonance (NMR) technique. Storage at −20 °C preserved telomerase activity in samples from cancer patients for at least 14 days (21.46 ± 0.135 versus 21.84 ± 0.357, p = .756), while samples obtained from healthy persons should be stored at −80 °C. We observed significant decrease of telomerase activity at freeze thaw cycle 5 in cancer patients’ samples (21.46 ± 0.135 versus 23.09 ± 0.316, p < .05), and in healthy persons’ ones already at cycle 3 (22.74 ± 0.107 versus 24.85 ± 0.151, p < .05). Telomerase activity from cell lines samples showed overall greater stability regarding the storage period and freeze-thaw cycles and it was considered for standard sample, which was confirmed by NMR analysis. Telomerase enzyme had adequate stability while efficacy, linearity, and reproducibility of TRAP method were acceptable for bio-analytical methods. All this indicated that telomerase could be a reliable biomarker.

Amplification-free and direct fluorometric determination of telomerase activity in cell lysates using chimeric DNA-templated silver nanoclusters.

A fluorogenic probe has been developed for determination of telomerase activity using chimeric DNA-templated silver nanoclusters (AgNCs). The formation of AgNCs was investigated before (route A) and after (route B) telomerase elongation reaction. Both routes caused selective quenching of the yellow emission of the AgNCs (best measured at excitation/emission wavelength of 470/557nm) in telomerase-positive samples. The quenching mechanism was studied using synthetically elongated DNA to mimic the telomerase-catalyzed elongation. The findings show that quenching is due to the formation of parallel G-quadruplexes with a -TTA- loop in the telomerase elongated products. The assay was validated using different cancer cell extracts, with intra- and interassay coefficients of variations of <9.8%. The limits of detection for MCF7, RPMI 2650 and HT29 cell lines are 15, 22 and 39 cells/μL. This represents a distinct improvement over the existing telomeric repeat amplification protocol (TRAP) assay in terms of time, sensitivity and cost. Graphical Abstract A method was developed using chimeric DNA-templated silver nanoclusters to detect telomerase activity directly in cell extracts. The sensitivity of this new method outperforms the traditional TRAP assay, and without the need for amplification.

Nonradioactive and Radioactive Telomerase Assays for Detecting Diminished Telomerase Activity in Cancer Cells after Treatment with Retinoid.

Detection of any decrease in telomerase activity in cancer cells and tumor tissues is an important part in assessing overall therapeutic outcomes of a treatment agent in the laboratory and clinical settings. Almost 85% of cancers have activation of telomerase activity that promotes cell proliferation and discourages differentiation to sustain growth of the cancers. Retinoids are highly regarded as the anti-proliferation and pro-differentiation agents that cause down regulation of telomerase activity in the cancer cells. Two (nonradioactive and radioactive) telomeric repeat amplification protocol (TRAP) assays are optimized and fully described for detection of the diminished or abolished telomerase activity in a very low amount of protein extracts from cancer cells after treatment with a natural retinoid or a synthetic retinoid. These highly optimized and improved nonradioactive and radioactive TRAP assays can also be used for determining the presence or absence of telomerase activity in a small amount of any tumor tissue. The results from these TRAP assays can also help decide appropriate therapeutic options for the cancers with or without telomerase activity.

Phenotypic and functional differences between senescent and aged murine microglia.

Microglia, the key innate immune cells in the brain, have been reported to drive brain aging and neurodegenerative disorders; however, few studies have analyzed microglial senescence and the impact of aging on the properties of microglia. In the present study, we characterized senescence- and aging-associated phenotypes of murine brain microglia using well-accepted markers, including telomere length, telomerase activity, expression of p16INK4a, p21, p53, senescence-associated β-galactosidase, and a senescence-associated secretory phenotype. Quantitative real-time polymerase chain reaction analysis and a Telomeric Repeat Amplification Protocol assay indicated shortened telomeres and increased telomerase activity in senescent microglia, whereas telomeres remained unaltered and telomerase activity was reduced in aged microglia. Senescent microglia upregulated p16INK4a, p21, and p53, whereas acutely isolated microglia from the aged brain only exhibited a modest upregulation of p16INK4a. Senescent microglia showed decreased proliferation, while it was unchanged in aged microglia. Furthermore, microglia at late passages strongly upregulated expression of the senescent marker senescence-associated β-galactosidase. Senescent and aged microglia exhibited differential activation profiles and altered responses to stimulation. We conclude that microglia from the aged mouse brain do not show typical senescent changes because their phenotype and functional response strongly differ from those of senescent microglia invitro.

Chidamide-induced ROS accumulation and miR-129-3p-dependent cell cycle arrest in non-small lung cancer cells

BackgroundEpigenetic therapy is a promising popular treatment modality for various cancers. Histone modification and miRNA should not be underestimated in lung cancer. This study aimed to investigate whether chidamide, a histone deacetylase inhibitor (HDACi), which inhibits telomerase activity and induces cell cycle arrest, influences ROS and miRNA production in non-small cell lung cancer (NSCLC) cells. MethodsH1355 and A549 were treated with chidamide. The analysis of DNA content was measured by FACSCalibur equipped with a 488 nm laser. H1355 cells were transfected with miR-129-3p mimic by Lipofectamine2000. Telomerase activity was performed on the telomeric repeat amplification protocol (TRAP) assay. Detection of thymidylate synthase (TS), p21, p53, pRB, and β-actin, were performed by western blot analysis. ResultsOur data showed that expression of TS, p21, and pRB were altered in the presence of chidamide by PCR and western blot. Using BrdU-incorporation analysis, we found that chidamide induced G1 arrest through the regulation of the TS gene by miR-129-3p. Chidamide was shown to suppress telomerase activity in the TRAP assay and reduced the expression of human telomerase reverse transcriptase (hTERT) by PCR and q-PCR in H1355 and A549 cells. Chidamide increased the generation of reactive oxygen species (ROS) by flow cytometry. N-acetyl cysteine (NAC), a ROS scavenger, attenuated chidamide-induced telomerase activity inhibition. ConclusionChidamide repressed telomerase activity through ROS accumulation and cell cycle arrest by miR-129-3p upregulation in both H1355 and A549 cells. This is the first study to demonstrate that chidamide induces miR-129-3p upregulation and ROS accumulation, leading to cell cycle arrest.

TERTpromoter mutations are associated with poor prognosis and cell immortalization in meningioma

Meningiomas are mostly benign tumors tending to progress to higher-grade lesions. Mutations in the telomerase reverse transcriptase (TERT) gene promoter are comparably rare in meningioma, but were recently suggested to predict risk of recurrence and progression. Here we have analyzed a cohort of World Health Organization grades I-III meningiomas regarding the impact of TERT promoter mutations on patient prognosis and in vitro cell propagation feasibility. From 110 meningioma patients, 128 tissue samples were analyzed for the TERT promoter mutations C228T and C250T by direct sequencing. Of the 128 samples, 121 were tested for cell propagation in vitro. Telomerase activity, TERT mRNA expression, and telomere lengths were investigated by telomeric repeat amplification protocol assay, reverse transcription PCR, and quantitative PCR, respectively. Impact of the E-twenty-six (ETS) transcription factor inhibitor YK-4-279 on cell viability and TERT promoter activity was analyzed. TERT promoter mutations were found in 5.5% of all samples analyzed and were associated with a significantly upregulated telomerase activity and TERT mRNA expression (P < 0.0001 both). Regarding telomere lengths, no significant difference between the TERT promoter wild-type and mutated subgroups was detected. Patients with TERT promoter mutated tumors exhibited significantly shorter overall survival (P = 0.0006; 53.8 vs 115.6 mo). The presence of TERT promoter mutations but not telomerase activity or TERT mRNA expression predicted indefinite cell growth in vitro. TERT promoter mutated meningioma cells were hypersensitive against the ETS transcription factor inhibitor YK-4-279, inducing a distinct downregulation of TERT promoter activity. TERT promoter mutations drive meningioma aggressiveness, resulting in reduced patient survival, but might also open novel therapeutic options for progressive disease.

Abstract 1993: Rapid clonal evolution of dormant micrometastatic disease in head and neck cancer

Abstract Head and neck cancer (HNC) is prone to locoregional recurrence which results in poor clinical outcome. Recent HNC clinical studies demonstrated that cervical lymph node dissection results in improved patient outcome even in early disease. These results suggest that dormant micrometastatic cancer occurs early in head and neck carcinogenesis. We used our established in vivo chemical carcinogenesis model of head and neck cancer to characterize dormant micrometastatic disease using genetic lineage tracing and null mutant mice. Histopathology was analyzed by hematoxylin and eosin staining. Telomere DNA damage response was determined by immunofluorescent 53BP1 localization at telomeres, and by ATM/ATR western blotting. Keratin expression and proliferation index were determined by immunohistochemistry. Apoptotic cells were determined by TUNEL analysis. Telomerase activity was determined by telomere repeat amplification protocol. Alternative lengthening of telomeres was determined by PML localization at telomeres, telomere circular DNA analysis, and chromosome orientation fluorescence in situ hybridization. Genomic instability was determined by high throughput sequencing. Cancer stem cells were sorted by flow cytometry and transplanted subcutaneously in Matrigel. Advanced HNC exhibited significant metastatic disease in the majority of lymph nodes. Early HNC and even premalignant lesions exhibited micrometastatic disease in most lymph nodes. These micrometastatic lesions contained multiple HNC clones that were dependent for proliferation on paracrine chemokine signaling from a tumorigenic keratin 15+ cancer stem cell population. In situ, the micrometastatic K15+ cancer stem cell population exhibited increased telomere DNA damage response including activated ATM and ATR which correlated with low proliferation index. Clonal evolution of K15+ micrometastatic cancer stem cells was dependent on extremely short telomeres. These cells expressed both telomerase and recombination-based alternative lengthening of telomeres (ALT) activities. K15+ micrometastatic cancer stem cells exhibited high levels of genomic instability resulting from telomere fusions and expressed metastasis-specific gene expression programs. These patterns of micrometastatic gene expression revealed targeted therapeutic vulnerabilities. These results indicate that dormant micrometastatic disease develops rapidly during head and neck carcinogenesis from normal epithelial cells with short telomeres and is sensitive to targeted therapies. Citation Format: Angelica M. Lagunas, Jianchun Wu, David L. Crowe. Rapid clonal evolution of dormant micrometastatic disease in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1993.

Abstract 1464: Telomere DNA damage links benign prostatic hypertrophy, intraepithelial neoplasia, and prostate cancer

Abstract Telomere DNA damage has been demonstrated in benign prostatic hypertrophy (BPH), which is associated with prostate epithelial cell senescence. Telomere DNA damage is the most frequently observed genetic alteration in prostatic intraepithelial neoplasia, and is associated with poor clinical outcomes in prostate cancer. Gene expression database analysis revealed decreased TRF2 expression during malignant progression of the prostate gland. We reasoned that reduced TRF2 expression in prostate epithelium, by activating the telomere DNA damage response, would allow us to model both benign and malignant prostate disease. TRF2 expression was deleted in prostate epithelium using conditional null mutant mice. TRF2 expression in prostate glands and tumors was determined by quantitative reverse transcription polymerase chain reaction. Histopathology was analyzed by hematoxylin and eosin staining. Telomere DNA damage response was determined by 53BP1 localization at telomeres, and by ATM/Chk2/p53 western blotting. Androgen receptor, smooth muscle actin, keratin expression, and proliferation index were determined by immunohistochemistry. Cellular senescence was determined by histone H3K9me3 immunofluorescence microscopy. Apoptotic cells were determined by TUNEL analysis. Telomerase activity was determined by telomere repeat amplification protocol. Alternative lengthening of telomeres was determined by PML localization at telomeres and telomere circular DNA analysis. Genomic instability was determined by metaphase telomere fluorescence in situ hybridization. Sca1+/CD49f+ prostate cancer stem cells were sorted by flow cytometry and transplanted subcutaneously with urogenital sinus mesenchyme cells in Matrigel. Prostate glands with reduced epithelial TRF2 expression developed age- and p53-dependent hypertrophy, senescence, ductal dilation, and smooth muscle hyperplasia similar to human BPH. Prostate tumors with reduced TRF2 expression were classified as high grade androgen receptor negative luminal adenocarcinomas which exhibited decreased latency, increased proliferation, and distant metastases. Prostate cancer stem cells with reduced TRF2 expression were highly tumorigenic and maintained telomeres both by telomerase and alternative lengthening (ALT). Telomerase inhibition in prostate tumors with reduced TRF2 expression produced significant reduction in prostate tumor incidence by halting progression at intraepithelial neoplasia (PIN). These lesions were highly differentiated, exhibited low proliferation index, and high apoptotic cell fraction. Prostate tumors with reduced TRF2 expression and telomerase inhibition failed to metastasize and did not exhibit ALT. Our results demonstrate that the telomere DNA damage response links BPH, PIN, and prostate cancer and may be therapeutically manipulated to prevent prostate cancer progression. Citation Format: Jianchun Wu, David L. Crowe. Telomere DNA damage links benign prostatic hypertrophy, intraepithelial neoplasia, and prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1464.

Telomerase reverse transcriptase suppression inhibits cell proliferation and promotes cell apoptosis in hepatocellular cancer.

We aimed to investigate the role of telomerase reverse transcriptase (TERT) in hepatocellular cancer (HCC) cells. R software was used for differential expressed gene analysis. Western blot and quantitative real time polymerase chain reaction (qRT-PCR), respectively, were used to detect protein expression and mRNA level of TERT in tumor cell lines. Real-time quantitative telomeric repeat amplification protocol assay, MTT assay, colony formation assay, and flow cytometry (FCM) assay were used to analyze the telomerase activity, viability, proliferation, cell cycle progression and apoptosis of HCC cells. The proliferation ratio of HCC cells transfected with TERT-siRNA was significantly decreased compared with control group. Plate clone results suggested that the number of colonies also decreased in TERT-siRNA group. FCM results showed that more cells were arrested in G0/G1 phase and apoptosis rate increased in TERT-siRNA group compared with control group. TERT suppression inhibited cell proliferation but promoted cell cycle arrest and cell apoptosis. © 2018 IUBMB Life, 70(7):642-648, 2018.

Telomerase Inhibition by a New Synthetic Derivative of the Aporphine Alkaloid Boldine.

Telomerase, the enzyme responsible for cell immortality, is an important target in anti-cancer drug discovery. Boldine, an abundant aporphine alkaloid of Peumus boldus, is known to inhibit telomerase at non-toxic concentrations. Cytotoxicity of N-benzylsecoboldine hydrochloride (BSB), a synthetic derivative of boldine, was determined using the MTT method in MCF7 and MDA-MB231 cells. Aliquots of cell lysates were incubated with various concentrations of BSB in qTRAP (quantitative telomere repeat amplification protocol)-ligand experiments before substrate elongation by telomerase or amplification by hot-start Taq polymerase. The crystal structure of TERT, the catalytic subunit of telomerase from Tribolium castaneum, was used for docking and molecular dynamics analysis. The qTRAP-ligand data gave an IC50 value of about 0.17 ± 0.1 µM for BSB, roughly 400 times stronger than boldine, while the LD50 in the cytotoxicity assays were 12.5 and 21.88 µM, respectively, in cells treated for 48 h. Although both compounds interacted well with the active site, MD analysis suggests a second binding site with which BSB interacts via two hydrogen bonds, much more strongly than boldine. Theoretical analyses also evaluated the IC50 for BSB as submicromolar. BSB, with greater hydrophobicity and flexibility than boldine, represents a promising structure to inhibit telomerase at non-toxic concentrations.

Activated carbon N-acetylcysteine microcapsule protects against nonalcoholic fatty liver disease in young rats via activating telomerase and inhibiting apoptosis.

Non-alcoholic fatty liver disease (NAFLD) is becoming one of the world's most common chronic liver diseases in childhood, yet no therapy is available that has been approved by the food and drug administration (FDA). Previous studies have reported that telomere and telomerase are involved the development and progression of NAFLD. This study was designed to investigate the potential beneficial effects of activated carbon N-acetylcysteine (ACNAC) microcapsules on the development of NAFLD in young rats as well as the underlying mechanism(s) involved. Three-week old male Sprague Dawley rats were given high-fat diet (HFD) with/without ACNAC treatment for 7 consecutive weeks. Liver pathologies were determined by hematoxylin and eosin (H&E) and Oil Red O staining, as well as by changes in biochemical parameters of plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels, respectively. Glucose homeostasis was evaluated by the glucose tolerance test and the liver telomere length and activity were measured by real time PCR and telomeric repeat amplification protocol (TRAP). Western blot analysis was performed to determine the expression level of Bcl-2, Bax and Caspase-3. Our results demonstrated that ACNAC supplementation improved liver pathologies of rats that received long-term HFD feeding. ACNAC supplementation prevented HFD-induced telomere shortening and improved telomerase activity. Moreover, in comparison to HFD-fed rats, ACNAC supplementation markedly increased the expression of Bcl-2, but significantly decreased the expression of Bax and Caspase-3 in juvenile rats. Together, these results indicate that ACNAC may be a promising choice for preventing and treating NAFLD among children.

DdTRAP: A Method for Sensitive and Precise Quantification of Telomerase Activity.

Telomerase is a cellular RNA template-dependent reverse transcriptase that adds telomere repeats to the 3' ends of chromosomes. Telomerase is expressed almost universally in tumor cells (>85%) to maintain telomere length, thus providing the ability of tumor cells to avoid senescence and to have unlimited replication ability, one of the key hallmarks of cancer. ddTRAP (droplet digital Telomere Repeat Amplification Protocol) is a two-step assay with whole cell lysates that utilizes a telomerase-mediated primer extension followed by droplet digital PCR (ddPCR) detection of extended products. The adoptation of the TRAP assay to ddPCR has resulted in improved throughput, increased sensitivity and better repeatability of the TRAP assay. The protocol described below details our procedures for ddTRAP.

ID: 1051 Telomerase activity, telomere length and P53 mutation detection on cellular senescence of Human Amnion Mesenchymal Stem Cells (HAMCs)

A fundamental understanding of senescence in human amnion mesenchymal stem cells (HAMCs) is crucial for its application in cellular therapy. Previous findings strongly support that HAMCs undergoes cellular senescence after long term in-vitro culture, with evidence of significant morphological changes and the presence of the senescent associated β-galactosidase (SA-β-Gal) marker. The telomere length and the telomerase activity have been linked with cellular aging and they are important in regulating cell proliferation. In addition, p53 gene has been associated with cell senescence. The aim of this study was to investigate the telomerase activity, telomere length in senescent HAMCs, and to detect p53 mutations in these cells. Samples were obtained from amnion placenta and then cultured for long term. Prolong-cultured HAMCs was isolated at passages 5, 10 and 15 and then analysed via telomeric repeat amplification protocol (TRAP), telomere length assay and p53 mutation detection assay. The results showed that after long term culture of HAMCs, there was a decrease in telomere length and telomerase activity from passages 5, 10 to 15. Telomerase controls the telomere’s length which maintains the cells proliferation. The decrease of telomere length and telomerase activity may suggest that the proliferation of HAMCs has slowed down due to HAMCs entering senescence after long term culture. P53 mutation detection study indicated that HAMCs at all passage did not have altered sequences. Thus, the cells did not undergo uncontrollable replication due to the effect of long-term culture. Further studies on senescence in HAMCs will be assessed by investigating the expression level of p53, p21, p16, pRB and GADD45 genes in long term culture of HAMCs via RT-qPCR. The findings will help us understand the associations between gene expressions and the process of senescence

High glucose and free fatty acids induce endothelial progenitor cell senescence via PGC-1α/SIRT1 signaling pathway.

The objective of the research was to investigate the function of endothelial progenitor cells (EPCs) in the conditions of high glucose and lipids, which has been widely used to mimic the metabolic disorder that occurs in type 2 diabetic mellitus, and further to verify the role of PGC-1α and SIRT1, cellular energy metabolism regulators, in the process of senescence of EPCs with these combined stimuli. Circulating EPCs were incubated in absence or presence of high glucose (25 mM), FFA (200 µM) or both. EPCs senescence was assessed by β-galactosidase staining, EPCs telomerase activity was measured by telomeric repeat ampli-fication protocol assay, in vitro angiogenesis assay and MTT assays were performed to assess angiogenesis and proliferation ability of EPCs. The results showed that combined stimuli inhibited EPCs reendothelialization ability in vitro, accelerated EPCs senescence and decreased the telomerase activity. Meanwhile, with combined stimuli, the expression of PGC-1α increased whereas SIRT1 expression decreased in EPCs accompanied by activation of P53/P21 signaling pathway. Conversely, transfection of EPCs with PGC-1α-siRNA rescued EPCs premature senescence and up-regulated SIRT1 and decreased P53/P21 expression, correlating closely with the down-regulation of PGC-1α itself. In addition, the combined stimuli induced up-regulation of PGC-1α expression was partly mediated by ROS and P38 signaling pathway. Overall, the data presented here identify PGC-1α as a potent negative regulator of EPCs' senescence under combined stimuli, which is partly mediated by SIRT1/P53/P21 signaling pathway.

Changes in telomerase activity due to alternative splicing of human telomerase reverse transcriptase in colorectal cancer.

Human telomerase reverse transcriptase (hTERT) expression level may not always correlate with telomerase activity. The present study analyzed hTERT splicing patterns with respect to hTERT and telomerase activity in colorectal cancer. Telomerase activity was determined by telomeric repeat amplification protocol assay, and spliced variants of hTERT were identified by reverse transcription-polymerase chain reaction in 40 colorectal cancer tissue samples. In the lower range of telomerase activity (0-100 units), the percentage of the β variant decreased with the increment in telomerase activity, whereas in the higher range of telomerase activity (>100 units), total hTERT expression level revealed a trend toward increment. There was a positive correlation between the full-length variant level and β variant level. Conversely, there was a negative correlation between the percentage of the full-length variant and β variant. Tumor-node-metastasis stage was the strongest prognostic factor in multivariate analysis and the percentage of the full-length variant was an independent prognostic factor for survival. Telomerase activity was primarily altered with changes in alternative splicing of the full-length and β variants of hTERT in colorectal cancer.

Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay

BackgroundNatural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. MethodsThe study focuses on the mechanism of telomerase inhibition by stabilization of telomeric G-quadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol (q-TRAP) and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric G-quadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids. Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. ResultsThe results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. ConclusionsWe report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex stabilization. General significanceUnderstanding structural and molecular mechanisms of anti-cancer agents can help in developing new and more potent drugs with fewer side effects. Isoquinolines are the most biologically active agents from Chelidonium majus, which have shown to be telomeric G-quadruplex stabilizers and potent telomerase inhibitors.

Arylsulfanyl groups - Suitable side chains for 5-substituted 1,10-phenanthroline and nickel complexes as G4 ligands and telomerase inhibitors

Guanine-rich DNA sequences can undergo self-assembly into unique G-quadruplex structures that interfere with the binding of proteins to the same DNA region. The formation of DNA G-quadruplexes requires monovalent cations (Na+ and K+) or small molecules known as G-quadruplex (G4) ligands. Phenanthroline is a type of G4 ligand scaffold known for its coordination with metal ions to form complexes with a large aromatic surface area, which aptly stack with G-quartets. In this report, we have investigated the side chain effect on G-quadruplex recognition by evaluating a series of 5-substituted phenanthroline-based metal complexes (Phen-Ni) binding to telomeric G-quadruplex DNA. Results from biophysical methods including fluorescence and circular dichroism (CD) thermal denaturation, CD titration, and the fluorescent intercalator displacement (FID) assay suggest that several Phen-Ni complexes bind to G-quadruplex DNA with submicromolar G4DC50 values. Arylsulfanyl groups at the 5 position of 1,10-phenanthroline are the best side chains regarding binding affinity and selectivity towards G-quadruplex DNA. Most of the G-quadruplex binding Phen-Ni complexes can inhibit telomerase activity in vitro as indicated by the telomeric repeat amplification protocol (TRAP) assay and such inhibition is clearly concentration dependent. Our results here provide a guidance of utilizing 5-substituted phenanthroline derivatives as a viable and facile approach to design novel G4 ligands.

Does telomerase activity have an effect on infertility in patients with endometriosis?

ObjectiveThis study aimed to investigate the role of telomerase activity in the development of endometriosis-related infertility by evaluation of the serum telomerase in eutopic and ectopic endometrial tissue. Study designEutopic endometrium, cystic wall/ovarian cortex, and venous blood were assessed in forty-seven patients. The following groups of patients were identified: females with endometriosis requiring surgical intervention and healthy control females. Patients with histopathologically confirmed endometriosis were further subdivided in the infertile (n=14) and fertile (n=17) groups. Patients who underwent hysterectomy and oophorectomy for benign gynecological conditions were enrolled in the healthy control group (n=16). Telomerase activity was evaluated with three-group, endometriosis-based and fertility-based designs. Analyses were performed regardless the menstrual cycle phase (Phase G), in proliferative (Phase P) (n=22) and secretory phases (Phase S) (n=25).Telomeric Repeat Amplification Protocol PCR was applied for telomerase activity assessment.All statistical analyses were performed with STATA 14.2, GraphPad Prisma 7.01. ResultsIn analyses of the eutopic endometrium, with three-group design, a significant difference was not found in Phase G and P (p=0.58 and p=0.33, respectively). However, a statistical difference was shown in Phase S (p=0.008). A significant difference was not established in Phase G, P and S of endometriosis-based design (p=0.35, p=1.0, p=0.13, respectively). No difference was detected in Phase G and P of fertility-based design (p=0.66 and p=0.14, respectively), whereas in secretory phase difference was approved (p=0,049).Telomerase activity was not established in ectopic endometrium and in serum assessment. ConclusionsTelomerase activity is useless as a biomarker in peripheric blood analysis. The absence of activity in cystic wall approves the high differentiation of endometriosis tissue, what is the possible reason of low malignancy risk. The high rate of telomerase activity in the eutopic endometrium of the infertile group may be considered as a cause of endometriosis-related infertility.

Methylation of CDKN2B CpG islands is associated with upregulated telomerase activity in children with acute lymphoblastic leukemia.

The aim of the present study was to investigate the association between methylation of cyclin-dependent kinase inhibitor 2B (CDKN2B) CpG islands and telomerase activity in children with acute lymphoblastic leukemia (ALL). A total of 72 children with ALL and 12 children with immune thrombocytopenia (ITP) were subjected to bone marrow aspiration and methylation-specific polymerase chain reaction analysis, and modified telomeric repeat amplification protocol assay analyses, to evaluate CDKN2B methylation and telomerase activity, respectively. The results of the present study demonstrated that, of these 72 children with ALL, 31 exhibited CDKN2B methylation at diagnosis (43.1%), whereas 41 exhibited no CDKN2B methylation (36.9%). However, no CDKN2B methylation was detected in the ITP controls. Furthermore, the mean level of telomerase activity was 39.52±39.33 total product generated (TPG) units in children with ALL, which was significantly increased compared with 2.49±2.27 TPG units in the ITP controls (P=0.002). The mean levels of telomerase were 49.09±44.43 and 29.99±32.43 TPG units in children with ALL with and without CDKN2B methylation, respectively (P=0.041), therefore children with ALL exhibited significantly increased levels of telomerase. The increased telomerase activity was significantly associated with increased risk of childhood ALL (P=0.023). A total of 22/31 children with ALL with methylated CDKN2B (71.0%) and 17/41 children with ALL with unmethylated CDKN2B (41.46%) exhibited increased telomerase activity (>15 TPG units). The results of the present study suggest that hypermethylation of CDKN2B CpG islands and hyperactivity of telomerase are common events in childhood ALL, and hypermethylation of CDKN2B CpG islands was significantly associated with upregulated telomerase activity (P=0.013).