Telomerase Repeat Amplification Protocol Assay Research Articles

LINC02454-CCT complex interaction is essential for telomerase activity and cell proliferation in head and neck squamous cell carcinoma

Telomerase activity is upregulated in head and neck squamous cell carcinoma (HNSCC), yet its regulatory mechanisms remain unclear. Here, we identified a cancer-specific lncRNA (LINC02454) associated with poor prognosis by using LncRNA chip of our HNSCC cohorts and external datasets. Through employing negative-stain transmission electron microscopy (NS-TEM), we discovered an interaction between LINC02454 and CCT complex which would augment telomerase activity for maintaining telomere homeostasis. Supporting this, in the telomerase repeat amplification protocol (TRAP) assay and quantitative fluorescence in situ hybridization (Q-FISH) analysis, LINC02454 depletion significantly reduced telomerase activity and shortened telomere length. Consistently, pathways related to telomerase, mitosis, and apoptosis were significantly impacted upon LINC02454 knockdown in RNAseq analysis. Functionally, LINC02454-deficient cells exhibited a more significant senescence phenotype in β-galactosidase staining, cell cycle, and apoptosis assays. We further confirmed the role of LINC02454 in HNSCC proliferation through a combination of in vitro and in vivo experiments. The therapeutic potential of targeting LINC02454 was verified by adenovirus-shRNA approach in HNSCC patient-derived xenograft (PDX) models. In summary, our findings provided valuable insights into the molecular mechanisms of HNSCC tumorigenesis and potential targets for future treatment modalities.

Antiproliferative and Antitelomerase Effects of Silymarin on Human Colorectal and Hepatocellular Carcinoma Cells.

Silymarin, a widely-used hepatoprotective agent, has shown antitumor properties in both in vitro and animal studies. Currently, there is limited knowledge regarding silymarin's antitelomerase effects on human colorectal cancer and hepatocyte carcinoma cells. In this study, we investigated the antiproliferative and antitelomerase effects of silymarin on four human colorectal cancer and HepG2 hepatocyte carcinoma cell lines. The cell viability and telomerase activity were assessed using MTT and the telomerase repeat amplification protocol assay, respectively. We also investigated the effects of silymarin on the expression of human telomerase reverse transcriptase and its promoter methylation in HepG2 cells by real-time RT-PCR and methylation-specific PCR, respectively. Silymarin treatment inhibited cell proliferation and telomerase activity in all cancer cells. After 24 h of treatment, silymarin exhibited IC50 values ranging from 19 - 56.3 µg/mL against these cancer cells. A 30-min treatment with silymarin at the IC50 concentration effectively inhibited telomerase activity in cell-free extracts of both colorectal cancer and hepatocyte carcinoma cells. Treatment of HepG2 cells with 10 and 30 µg/mL of silymarin for 48 h resulted in a decrease in human telomerase reverse transcriptase expression to 75 and 35% of the level observed in the untreated control (p < 0.01), respectively. Treatment with silymarin (10, 30, and 60 µg/mL) for 48 h did not affect human telomerase reverse transcriptase promoter methylation in HepG2 cells. In conclusion, our findings suggest that silymarin inhibits cancer cell growth by directly inhibiting telomerase activity and downregulating its human telomerase reverse transcriptase catalytic subunit. However, silymarin did not affect human telomerase reverse transcriptase promoter methylation at the concentrations of 10 - 60 µg/mL used in this study.

Measuring telomerase activity using TRAP assays.

Telomerase is a reverse transcriptase that consists of the telomerase reverse transcriptase (TERT) protein and the telomerase RNA component TERC which also harbors the template region for telomere synthesis. In its canonical function the enzyme adds single-stranded telomeric hexanucleotides de novo to the ends of linear chromosomes, telomeres, in telomerase-positive cells such as germline, stem- and cancer cells. This potential biochemical activity of telomerase can be measured with the help of a telomerase repeat amplification protocol (TRAP) which often includes a PCR amplification due to the low abundance of telomerase in most cells and tissues. The current chapter describes various TRAP methods to detect telomerase activity (TA) using gel-based methods, its advantages and deficits, how to perform an ELISA-based TRAP assay and how best to interpret its results. Since development of the TRAP assay in 1994, there have been numerous modifications and adaptations of the method from real-time PCR analysis, isothermal amplification and nanotechnology to CRISPR/Cas-based methods which will be briefly mentioned. However, it is not possible to cover all different TRAP methods and thus there is no comprehensiveness claimed by this chapter. Instead, the author describes various aspects of using TRAP assays including required controls, sample preparation, etc. in order to avoid pitfalls and set-backs in applying this rather complex and demanding technique. The TRAP assay is particularly important to support clinical diagnosis of cancer, analyze tumor therapy as well as to evaluate various approaches to inhibit TA as a form of anti-cancer therapy.

Teloxantron inhibits the processivity of telomerase with preferential DNA damage on telomeres

Telomerase reactivation is one of the hallmarks of cancer, which plays an important role in cellular immortalization and the development and progression of the tumor. Chemical telomerase inhibitors have been shown to trigger replicative senescence and apoptotic cell death both in vitro and in vivo. Due to its upregulation in various cancers, telomerase is considered a potential target in cancer therapy. In this study, we identified potent, small-molecule telomerase inhibitors using a telomerase repeat amplification protocol assay. The results of the assay are the first evidence of telomerase inhibition by anthraquinone derivatives that do not exhibit G-quadruplex-stabilizing properties. The stability of telomerase in the presence of its inhibitor was evaluated under nearly physiological conditions using a cellular thermal shift assay. Our data showed that the compound induced aggregation of the catalytic subunit (hTERT) of human telomerase, and molecular studies confirmed the binding of the hit compound with the active site of the enzyme. The ability of new derivatives to activate DNA double-strand breaks (DSBs) was determined by high-resolution microscopy and flow cytometry in tumor cell lines differing in telomere elongation mechanism. The compounds triggered DSBs in TERT-positive A549 and H460 lung cancer cell lines, but not in TERT-negative NHBE normal human bronchial epithelial and ALT-positive U2OS osteosarcoma cell lines, which indicates that the induction of DSBs was dependent on telomerase inhibition. The observed DNA damage activated DNA damage response pathways involving ATM/Chk2 and ATR/Chk1 cascades. Additionally, the compounds induced apoptotic cell death through extrinsic and intrinsic pathways in lung cancer cells. Taken together, our study demonstrated that anthraquinone derivatives can be further developed into novel telomerase-related anticancer agents.

Telomerase Activity Analysis In Urine Sediment for Bladder Cancer.

Bladder cancer with an incidence of 15 cases per 100,000 persons in the global population is the most common tumor of the urinary tract. Imaging techniques, cytoscopy, and cytology are not sufficiently accurate to detect early stage tumors, and the need for new diagnostic markers is still an urgency. Among the biomarkers most recently proposed to improve diagnostic accuracy and especially sensitivity, increasing attention has been focused on the role of the ribonucleoprotein, telomerase. Previous studies have shown that the quantitative telomerase repeat amplification protocol (TRAP) assay performed in voided urine is an important noninvasive tool for the diagnosis of bladder tumors since it has very high sensitivity and specificity, even for early stage and low-grade tumors. Telomerase activity in urine determined by TRAP seems to be marker of great potential, even more advantageous in cost-benefit terms when used in selected symptomatic patients or professionally high-risk subgroups. Here we report the real-time PCR protocol to detect telomerase activity in urine sediment for bladder cancer.

Cost-Effective Trap qPCR Approach to Evaluate Telomerase Activity: an Important Tool for Aging, Cancer, and Chronic Disease Research

OBJECTIVES:Telomeres are a terminal “DNA cap” that prevent chromosomal fusion and degradation. However, aging is inherent to life, and so is the loss of terminal sequences. Telomerase is a specialized reverse transcriptase encoded by self-splicing introns that counteract chromosome erosion. Telomerase activity is observed during early embryonic development, but after the blastocyst stage, the expression of telomerase reduces. The consequences of either insufficient or unrestrained telomerase activity underscore the importance of ongoing studies aimed at elucidating the regulation of telomerase activity in humans. In the present study, we aimed to standardize a simplified telomerase repeat-amplification protocol (TRAP) assay to detect telomerase activity in unstimulated and PHA-stimulated mononuclear cells.METHODS and RESULTS:Our optimized qPCR-based can efficiently evaluate telomerase activity. Quantification of protein and DNA between unstimulated and PHA-stimulated peripheral blood mononuclear cells revealed cellular activation and cell-cycle entry. The assay also showed that relative telomerase activity is significantly different between these two conditions, supporting the applicability of the assay. Furthermore, our findings corroborated that telomerase activity decreases with age.CONCLUSIONS:Telomeres and telomerase are implicated in aging and development of chronic diseases and cancer; however, difficulty in accessing commercial kits to investigate these aspects is a critical constraint in health surveillance studies. Our optimized assay was successfully used to differentiate telomerase activity between unstimulated and stimulated cells, clearly showing the reactivation of telomerase upon cell activation. This assay is affordable, reproducible, and can be executed in resource-limited settings.

PO-282 SMAR1 transcriptionally repress hTERT and alters the stem cell population in colorectal cancer cells

IntroductionOne of the distinguishing features that set the cancer cell apart is its ability for indefinite proliferation. Cancer progression is accompanied by perturbations in the chromatin structure that results in inappropriate gene expression and genomic instability. Aberrant activation of signalling pathways in cancer cells lead to a cascade of events resulting in ‘replicative immortality’. One of the mechanisms by which cancer cells keep dividing is by the reactivation of enzyme telomerase (hTERT). hTERT reactivation confers tumour cell with various advantages like protection against DNA damaging agents, maintenance of stem cell pool, increased angiogenesis, etc. This cancer stem cell (CSC) population then remains quiescent owing to its radio and chemo-resistant nature and leads to cancer relapse.Material and methodsWestern Blotting, Real time PCR, Co-Immunoprecipitation, Chromatin Immunoprecipitation, Telomerase Repeat Amplification Protocol (TRAP) Assay, Polyps generation in Balb/c mice.Results and discussionsOur study elucidates the role of a Martix Attachment Region Binding Protein (MARBP), SMAR1 as a transcriptional repressor of hTERT. SMAR1 directly interacts with HDAC1/mSin3a co-repressor complex and bind to the hTERT promoter. This binding of SMAR1 brings about Wnt/β-Catenin mediated repression of hTERT. Interestingly we find that occupancy of HDAC1 on hTERT promoter is SMAR1-dependent. The recruitment of repressor complex on the hTERT promoter results in the deacetylation of histones and an increase in the H3K9me2 mark.This helps maintain a repressive environment on the hTERT promoter and alters the occupancy of PolII on the promoter. We also find that knock-down of SMAR1 promotes CD133+ CSC’s and SMAR1 over-expression inhibits CD133+ CSC’s. Moreover, CD133high and CD133low populations exhibit differential SMAR1 and hTERT expression. We generated polyps in Balb/c mice using azoxymethane (AOM)/dextran sodium sulphate (DSS). When the levels of SMAR1 and hTERT were assessed; polyps expressed lower levels of SMAR1 and elevated hTERT as compared to the adjacent non-cancerous tissue.ConclusionWe show that SMAR1 transcriptionally repress hTERT. Given that hTERT reactivation is central for tumour development, progression and cancer stem cell maintenance, this regulation by SMAR1 represents an important mechanism of tumour suppression and inhibition of cancer stem cell trait.

Ferrocenyl naphthalene diimides as tetraplex DNA binders

Seven ferrocenyl naphthalene diimide (FND) ligands were synthesized. Each had a higher affinity for tetraplex DNA than for either single- or double-stranded DNA. The FND binding affinities were >105M−1 in 0.10M AcOH-AcONa or AcOH-AcOK (pH5.5) containing 0.10M NaCl or KCl. The FNDs with the highest binding affinities for tetraplex DNA showed 23- or 8-times higher preference for tetraplex DNA than for single- or double-stranded DNA, respectively. The current signals generated from the seven FNDs bound to the tetraplex DNA immobilized on the electrode were found to correlate with the binding affinities of these ligands for the tetraplex DNA. Furthermore, using the telomerase repeat amplification protocol assay, the FND ligands could be categorized into three groups: (a) inhibiting both telomerase and Taq polymerase, (b) inhibiting telomerase alone, and (c) inhibiting neither telomerase nor Taq polymerase.

Role of diallyl disulfide-mediated cleavage of c-Myc and Sp-1 in the regulation of telomerase activity in human lymphoma cell line U937

ObjectiveGarlic (Allium sativum) has been considered a wonder herb for years with a reputation of disease prevention. Telomerase, a ribonucleoprotein enzyme responsible for telomere integrity, is strongly up-regulated in different types of cancers. The aim of this study was to reveal the role of diallyl disulfide (DADS), an organosulfur component of garlic, on telomerase activity in human lymphoma with an emphasis on key transcription factors c-Myc and Sp-1. MethodsHuman lymphoma cell line U937 was used as model cell line. Telomerase activity was measured by telomerase repeat amplification protocol assay, levels of related proteins and mRNAs were measured by Western blot and reverse transcriptase polymerase chain reaction, respectively. Moreover, in vitro binding assay was performed using radiolabeled double-stranded DNA having specific sequences to detect involvement of transcription factors in DADS-dependent modulation of telomerase activity. ResultsThe present study demonstrated DADS-mediated decrease in telomerase activity in U937 cells with concomitant transcriptional down-regulation of human telomerase reverse transcriptase (hTERT) that is caused by reduced binding of c-Myc and Sp-1 to their respective binding sites on hTERT promoter. Lowering of DNA-binding activity of c-Myc and Sp-1 due to DADS treatment is caused by the deactivation of these transcription factors due to cleavage. Additionally, Mad1—the repressor protein of hTERT expression—is also overexpressed in DADS-treated U937 cells. ConclusionsThese findings strongly suggest that DADS down-regulate telomerase activity through c-Myc-, Sp-1-, and Mad1-dependent transcriptional down-regulation of hTERT.

Stabilization of G-quadruplex DNA and inhibition of telomerase activity studies of ruthenium(II) complexes

Two ruthenium(II) complexes [Ru(IP)2(PIP)](ClO4)2·2H2O (1) and [Ru(PIP)2(IP)](ClO4)2·2H2O (2) (IP=imidazole [4, 5-f] [1,10] phenanthroline, PIP=2-phenylimidazo-[4, 5-f][1,10] phenanthroline) have been synthesized and characterized. The quadruplex binding of the compounds was evaluated by emission spectrum, CD spectroscopy, Visual detection assay and FRET (fluorescence resonance energy transfer)-melting assay. The results show that both complexes can induce the stabilization of quadruplex DNA, while complex 1 is a better G-quadruplex binder than complex 2. Furthermore, polymerase chain reaction-stop assay, electrophoretic mobility shift assay, telomerase repeat amplification protocol and MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay demonstrate that complex 1 not only can stabilize dimer forms of the G-quadruplex at low concentrations but also exhibit better inhibitory activity for telomerase and cancer cells.

Effects of gamma irradiation on cell cycle, apoptosis and telomerase activity in p53 wild-type and deficient HCT116 colon cancer cell lines

Radiotherapy serves as adjunctive treatment to chemotherapy and surgical resection of colorectal cancer. However, the cellular response to irradiation varies depending on the expression of tumor suppressor p53, which plays a significant role in the regulation of cell cycle arrest, apoptosis and telomerase activity in various cancers. The present study aimed to investigate cell cycle arrest, apoptosis and telomerase activity with respect to p53 expression in p53 wild-type (+/+) and deficient (−/−) HCT116 colon cancer cell lines following 5 Gy γ-irradiation. Cell cycle arrest and apoptosis were evaluated using flow cytometry. The telomerase activity was measured using a TRAP (telomerase repeat amplification protocol) assay. Following treatment with irradiation, G1/S cell cycle arrest occurred in the p53+/+ cells, whereas the p53−/− cells accumulated in the G2 phase. No differences were observed in the apoptotic ratios between the two cell lines following irradiation. Decreased telomerase activity was observed in the p53+/+ cells, whereas telomerase activity was increased in the p53−/− cells. The results showed that while telomerase activity and G1 cell cycle arrest were regulated depending on the p53 status, G2 arrest and the apoptotic response were promoted via a p53-independent pathway.

Investigation of telomerase activity in inflammatory and noninflammatory breast cancer.

e21055 Background: Inflammatory breast cancer (IBC) is an aggressive type of breast cancer disease that has a high incidence in Egypt than western countries. It is characterized by rapid progression, involvement of dermal lymphatic emboli and extensive lymph node involvement. Basic and translational studies needed to define IBC disease biology and identify specific biomarkers have been limited by the paucity of patient samples. Hence, the current study aimed to introduce the telomerase activity level as a novel diagnostic marker for breast cancer and specifically for IBC to be differentiated from non-IBC. Methods: Breast cancer patients were enrolled from Ain Shams University hospitals in Cairo, divided into two groups: IBC (n=26) and non-IBC (n=27). Tissue samples were collected during modified radical mastectomy. TRAP (Telomerase repeat amplification protocol) assay was used to assess the telomerase activity in inflammatory and non inflammatory breast cancer tissue samples and their representative cell lines, SUM149 and MCF-7 respectively. Immunohistochemistry was used to investigate the expression of hTERT subunit of telomerase in paraffin embedded tissue samples of both types of patients. MTT assay was used to compare the effect of AZT (3’-azido-3’-deoxythymidine) on the growth of cell lines SUM149 and MCF-7. Results: : IBC showed Telomerase activity ranged from 12 to 376 units with a mean value of 75 and a median value of 43, while telomerase activity in non-IBC ranged from 6 to 109 units with a mean value of 35 and a median value of 24. On the other hand, normal tissues showed telomerase activity below 5 (P &gt; 0.001). Using immunohistochemistry, the hTERT expression was higher in IBC than non-IBC and no expression at all in normal tissues. Also, a positive correlation was found between the telomerase activity and the number of metastatic lymph nodes in both IBC (r =0.511) and non-IBC (r =0.622). Besides, IBC cell line showed a faster response (53%) to telomerase inhibition by AZT after 24 hrs than non-IBC cell line (13%) at the same conditions. Conclusions: Telomerase could be a promising marker at the diagnostic and therapeutic levels in breast cancer and specifically in IBC.

Complementary treatment of siTERT for improving the antitumor effect of TERT-specific I-131 therapy

Sodium iodide symporter (NIS)-based radionuclide therapy provides an effective means of treating malignant tumors. However, it is sometimes inadequate because of limited effects on radio-resistant tumors, and thus, combination therapies with other therapeutic options have been requested to enhance its efficacy. Human telomerase reverse transcriptase (hTERT) has been reported to be involved in the progression of most cancers and also been viewed as a good candidate for targeting tumor. Application of TERT-specific radionuclide therapies using NIS gene transfer have been reported to treat TERT-positive tumors, but this approach only demonstrated tumor regression rather than eradication. As inhibiting TERT expression by introducing the hTERT-specific shRNA (siTERT) has been suggested as a therapeutic option, we investigated the complementary role of siTERT treatment after the TERT-specific I-131 therapy and its possibility as a novel anticancer therapeutic strategy. Retroviruses containing TERT promoter/NIS for TERT specific Radionuclide therapy and siTERT for TERT targeting antisense therapy were produced. Hep3B cells expressing TERT specific NIS (Hep3B-TERT/NIS) were xenografted into nude mouse and visualized with micro-SPECT/CT for monitoring NIS activity. The levels of hTERT mRNA, protein and its activity were confirmed by RT-PCR, Western blotting and Telomerase repeat amplification protocol assay. Cell proliferation was monitored by MTT assay and induced apoptosis was confirmed by Annexin-V-PI staining. Therapeutic effects of I-131 and/or siTERT were evaluated by clonogenic assay and mouse tumor model. Reduction of hTERT mRNA, protein and TERT activity by siTERT were observed in Hep3B-TERT/NIS cells. The viabilities of the infected cells were significantly decreased to 50% versus siScramble treated controls. The early apoptotic cell population was increased by siTERT. The survival rates of cells treated with siTERT or I-131 alone were 72.4±7.6% and 56.2±5.2%, respectively. However, the survival rate of cells treated with I-131 and siTERT were decreased to 22.1±2.8%. From mouse xenograft model, we also found that the siTERT gene therapy showed synergism to the radioiodine therapy for reducing tumor growth in vivo. Our Results suggested that complementary siTERT gene therapy offers a novel strategy of cancer therapy to improve the therapeutic efficacy of TERT-specific I-131.

Investigation of Telomerase Activity in Inflammatory and Non Inflammatory Breast Cancer

Background: Inflammatory breast cancer (IBC) is an aggressive type of breast cancer disease that has a high incidence in Egypt than western countries. It is characterized by rapid progression, involvement of dermal lymphatic emboli and extensive lymph node involvement. Basic and translational studies are needed to define IBC disease biology and identify specific biomarkers have been limited by the paucity of patient samples. Hence, the current study aimed to introduce the telomerase activity level as a novel diagnostic marker for breast cancer and specifically for IBC to be differentiated from non-IBC. Methods: Breast cancer patients were enrolled from Ain Shams University hospitals in Cairo, divided into two groups: IBC (n=26) and non-IBC (n=27). Tissue samples were collected during modified radical mastectomy. TRAP (Telomerase repeat amplification protocol) assay was used to assess the telomerase activity in inflammatory and non inflammatory breast cancer tissue samples. Immunohistochemistry was used to investigate the expression of hTERT subunit of telomerase in paraffin embedded tissue samples of both types of patients. Results: IBC showed Telomerase activity ranged from 12.2 to 367.1 units with a mean value of 78 and a median value of 43, while telomerase activity in non-IBC ranged from 6.1 to 109.34 units with a mean value of 41.1 and a median value of 24. On the other hand, normal tissues showed telomerase activity below 5 (P<0.001). Using immunohistochemistry, the hTERT expression was higher in IBC than non-IBC and no expression at all in normal tissues. Moreover, a positive mild correlation was found between the telomerase activity and the number of metastatic lymph nodes in both IBC (r=0.53) and non-IBC (r=0.54). Conclusions: Telomerase could be a promising marker at the diagnostic and therapeutic levels in breast cancer and specifically in IBC.

Vorinostat, SAHA, represses telomerase activity via epigenetic regulation of telomerase reverse transcriptase in non-small cell lung cancer cells

Vorinostat (suberoylanilide hydroxamic acid), a class of histone deacetylase inhibitors, represents an emerging class of anticancer agents currently progressing in clinical trials. It causes cell growth inhibition, differentiation, and apoptosis of many tumor types in vitro and in vivo. Recently, it was reported that hTERT is one of the targets for cancer therapy in cancer cells. Telomerase repeat amplification protocol assay was used to analyze the expression of hTERT after vorinostat treatment in the A549 lung cancer cells. Vorinostat inhibited telomerase activity by reducing the expression of human telomerase reverse transcriptase (hTERT) in A549 human lung cancer cells. The epigenetic regulation mechanism is responsible for the repression of hTERT by vorinostat, analyzed through the methylation-specific PCR and bisulfite sequencing of the hTERT promoter. Vorinostat induced the demethylation of site-specific CpGs on the promoter region of hTERT, which was caused by the down-regulation of DNA methyltransferases. DNA methyltransferases (DNMT1 and DNMT3b) were also decreased in vorinostat-treated A549 cancer cells. Furthermore, chromatin immunoprecipitation analysis of the hTERT promoter revealed that vorinostat decreased the level of inactive chromatin markers dimethyl-H3K9, and the declined binding of DNMT1 and DNMT3b were associated. The novel insights showed that vorinostat down-regulated telomerase via epigenetic alteration in lung cancer to vorinostat-mediated cancer-specific therapies.

Curcumin Regulates Low-Linear Energy Transfer γ-Radiation-Induced NFκB-Dependent Telomerase Activity in Human Neuroblastoma Cells

We recently reported that curcumin attenuates ionizing radiation (IR)-induced survival signaling and proliferation in human neuroblastoma cells. Also, in the endothelial system, we have demonstrated that NFκB regulates IR-induced telomerase activity (TA). Accordingly, we investigated the effect of curcumin in inhibiting IR-induced NFκB-dependent hTERT transcription, TA, and cell survival in neuroblastoma cells.SK-N-MC or SH-SY5Y cells exposed to IR and treated with curcumin (10-100 nM) with or without IR were harvested after 1 h through 24 h. NFκB-dependent regulation was investigated either by luciferase reporter assays using pNFκB-, pGL3-354-, pGL3-347-, or pUSE-IκBα-Luc, p50/p65, or RelA siRNA-transfected cells. NFκB activity was analyzed using an electrophoretic mobility shift assay and hTERT expression using the quantitative polymerase chain reaction. TA was determined using the telomerase repeat amplification protocol assay and cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide and clonogenic assay.Curcumin profoundly inhibited IR-induced NFκB. Consequently, curcumin significantly inhibited IR-induced TA and hTERT mRNA at all points investigated. Furthermore, IR-induced TA is regulated at the transcriptional level by triggering telomerase reverse transcriptase (TERT) promoter activation. Moreover, NFκB becomes functionally activated after IR and mediates TA upregulation by binding to the κB-binding region in the promoter region of the TERT gene. Consistently, elimination of the NFκB-recognition site on the telomerase promoter or inhibition of NFκB by the IκBα mutant compromises IR-induced telomerase promoter activation. Significantly, curcumin inhibited IR-induced TERT transcription. Consequently, curcumin inhibited hTERT mRNA and TA in NFκB overexpressed cells. Furthermore, curcumin enhanced the IR-induced inhibition of cell survival.These results strongly suggest that curcumin inhibits IR-induced TA in an NFκB dependent manner in human neuroblastoma cells.

Abstract 2740: Increased accuracy of TRAP-FISH combination in bladder cancer diagnosis in symptomatic patients

Abstract Introduction: Previous case-control studies have shown the importance of urine telomerase activity determined by the telomerase repeat amplification protocol (TRAP) assay to detect early bladder cancer. However, its validation is needed in large and consecutive series of symptomatic patients before it can be considered for routine clinical use. The aim of the present study was to verify whether a multiple approach based on first-level TRAP assay analysis and successive evaluation with UroVysion test, limited to patients with TRAP-positive urine samples, could unmask false-positive results and improve diagnostic specificity. Patients and methods: Three hundred individuals with urinary tract symptomatology (50 females, 250 males), but without prior history of bladder malignancy were consecutively enrolled between January 2007 and June 2008. All urine samples were processed for cytological examination and also for telomerase activity determination using a quantitative TRAP assay. FISH analysis was carried out only in TRAP-positive samples. Results: At a cut-off of 50 arbitrary enzymatic units (AEUs), shown in previous case control-studies as the most accurate diagnostic discriminant, the positive predictive value (PPV) and negative predictive value (NPV) using TRAP assay were 48% and 86%, respectively, with an overall accuracy of 71%. With the two-step TRAP-FISH approach, PPV increased to 75% and overall accuracy to 83%. Conclusion: Our results indicate that TRAP assay by itself could make a valid contribution to early bladder cancer diagnosis in high-risk patients with urinary tract symptomatology, and that its combination with FISH could improve specificity by unmasking false-positive TRAP results. *Contributed equally to this work Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2740.

Isolation and characterization of stem cells from the human parathyroid gland

Somatic stem cells can be obtained from a variety of adult human tissues. However, it was not clear whether human parathyroid glands, which secrete parathyroid hormones and are essential in maintaining homeostasis levels of calcium ions in the circulation, contained stem cells. We aimed to investigate the possibility of isolating such parathyroid-derived stem cells (PDSC). Surgically removed parathyroid glands were obtained with informed consent. Cell cytogenetics was used to observe chromosomal abnormalities. Surface phenotypes were characterized by flow cytometry. Telomerase repeat amplification protocol (TRAP) assay was performed to observe the telomerase activity. RT-PCR and real-time PCR was was used to detect gene expressions. Real-time calcium uptake imaging was performed for extent of calcium uptake and transmission electron microscopy and immunofluorecent staining for smooth muscle actin. After enzymatic digestion and primary culture, plastic-adherent, fibroblast-like cells appeared in culture and a morphologically homogeneous population was derived from subsequent limiting dilution and clonal expansion. Karyotyping was normal and doubling time of clonal cell growth was estimated to be 70.7 +/- 14.5 h (mean +/- standard deviation). The surface phenotype of the cells was positive for CD73, CD166, CD29, CD49a, CD49b, CD49d, CD44, CD105, and MHC class I, and negative for CD34, CD133, CD117, CD114, CD31, CD62P, EGF-R, ICAM-3, CD26, CXCR4, CD106, CD90 and MHC class II, similar to mesenchymal stem cells (MSC). Detectable levels of telomerase activity along with pluripotency Sall4 gene expression were observed from the isolated PDSCs. Expression of calcium-sensing receptor gene along with alpha-smooth muscle actin was induced and cellular uptake of extracellular calcium ions was observed. Furthermore, PDSCs possessed osteogenic, chondrogenic and adipogenic differentiation potentials. Our results reveal that PDSCs were similar phenotypically to MSCs and further studies are needed to formulate induction conditions to differentiate PDSCs into parathyroid hormone-secreting chief cells.

Synthesis and biological evaluation of novel 4,5-bis(dialkylaminoalkyl)-substituted acridines as potent telomeric G-quadruplex ligands

Several 4,5-bis(dialkylaminoalkyl)-substituted acridines have been prepared starting from acridine and their telomeric G-quadruplex stabilizing properties were evaluated using FRET melting and TRAP (Telomerase Repeat Amplification Protocol Assay) experiments.

The current role of telomerase in the diagnosis of bladder cancer

Bladder cancer has an incidence of 15 cases per 100,000 persons in the global population and is the most common tumor of the urinary tract. Imaging techniques, cytoscopy, and cytology are either invasive or not sufficiently accurate to detect early stage tumors, and the need for new diagnostic markers still remains. Among the markers most recently proposed to improve diagnostic accuracy and especially sensitivity, increasing attention has been focused on the role of the ribonucleoprotein, telomerase. Relevant papers on the etiology, diagnosis, and evaluation of bladder cancer using telomerase in urine were searched for and considered. The PubMed search was performed using the text terms “bladder cancer”, “diagnosis”, and “telomerase”. Previous studies have shown that the quantitative Telomerase Repeat Amplification Protocol (TRAP) assay performed in voided urine is an important non-invasive tool for the diagnosis of bladder tumors since it has very high sensitivity and specificity, even for early stage and low grade tumors. The main limitation of this test is the rate of false positive results due to the presence of inflammatory or non-tumor cells (i.e., epithelial cells from the lower genital tract), which express telomerase activity (TA). Consequently, an in situ analysis would seem to be important to identify the nature of telomerase-positive cells. Immunocytochemical detection of the hTERT subunit by a specific antibody seemed to open up the possibility to identify different cellular components of urine. However, the lack of a strict relationship between hTERT protein expression and telomerase activity has, to a certain extent, made this approach less relevant. In conclusion, telomerase activity in urine determined by TRAP seems to be marker of great potential, even more advantageous in cost/benefit terms when used in selected symptomatic patients or professionally high-risk subgroups.