Telomerase Activity In Cancer Cells Research Articles

NOVA1 directs PTBP1 to hTERT pre-mRNA and promotes telomerase activity in cancer cells

Alternative splicing is dysregulated in cancer cells, driving the production of isoforms that allow tumor cells to survive and continuously proliferate. Part of the reactivation of telomerase involves the splicing of hTERT transcripts to produce full-length (FL) TERT. Very few splicing factors to date have been described to interact with hTERT and promote the production of FL TERT. We recently described one such splicing factor, NOVA1, that acts as an enhancer of FL hTERT splicing, increases telomerase activity, and promotes telomere maintenance in cancer cells. NOVA1 is expressed primarily in neurons and is involved in neurogenesis. In the present studies, we describe that polypyrimidine-tract binding proteins (PTBPs), which are also typically involved in neurogenesis, are also participating in the splicing of hTERT to FL in cancer. Knockdown experiments of PTBP1 in cancer cells indicate that PTBP1 reduces hTERT FL splicing and telomerase activity. Stable knockdown of PTBP1 results in progressively shortened telomere length in H1299 and H920 lung cancer cells. RNA pulldown experiments reveal that PTBP1 interacts with hTERT pre-mRNA in a NOVA1 dependent fashion. Knockdown of PTBP1 increases the expression of PTBP2 which also interacts with NOVA1, potentially preventing the association of NOVA1 with hTERT pre-mRNA. These new data highlight that splicing in cancer cells is regulated by competition for splice sites and that combinations of splicing factors interact at cis regulatory sites on pre-mRNA transcripts. By employing hTERT as a model gene, we show the coordination of the splicing factors NOVA1 and PTBP1 in cancer by regulating telomerase that is expressed in the vast majority of cancer cell types.

Multivalent Self-Assembled DNA Polymer for Tumor-Targeted Delivery and Live Cell Imaging of Telomerase Activity.

The efficient detection and in situ monitoring of telomerase activity is of great importance for cancer diagnosis and biomedical research. Here we report for the first time that the development of a novel multivalent self-assembled DNA polymer, constructed through telomerase primer sequence (ITS) triggered hybridization chain assembly using two functional hairpin probes (tumor-trageting aptamer modified H1 and signal probe modified H2), for sensitive detection and imaging of telomerase activity in living cells. After internalizing into the tumor cells by multivalent aptamer targeting, the ITS on DNA polymers can be elongated by intracellular telomerase to generate telomere repeat sequences that are complementary with the signal probe, which can proceed along the DNA polymers, and gradually light up the whole DNA polymers, leading to an enhanced fluorescence signal directly correlated with the activity of telomerase. Our results demonstrated that the developed DNA polymer show excellent performance for specifically detecting telomerase activity in cancer cells, dynamically monitoring the activity change of telomerase in response to telomerase-based drugs, and efficiently distinguishing cancer cells from normal cells. The proposed strategy may afford a valuable tool for the monitoring of telomerase activity in living cells and have great implications for biological and diagnostic applications.

Sensitive detection of telomerase activity in cancer cells using portable pH meter as readout

This study developed a portable and sensitive biosensor for the detection of telomerase activity extracted from HeLa cells by monitoring urease regulated pH change. In the presence of telomerase, the telomerase substrate (TS) primer was elongated by adding telomeric repeats (TTAGGG)n firstly. The TS primer was then immobilized onto streptavidin magnesphere paramagnetic particles (PMPs). The elongated repeat unit can hybridize with biotinylated complementary DNA (cDNA) to specifically capture biotinylated urease onto PMPs. After magnetic separation, the PMPs complex was mixed with urea solution. As urease can catalyze the hydrolysis of urea into ammonia and induce pH increasing of the solution, the activity of telomerase can be detected by pH-sensitive dyes or by a portable pH meter easily. The changed pH has a linear relationship with the logarithm of the number of HeLa cells from 50 to 10,000 and with a detection limit of ~20 HeLa cells, which indicated the proposed method have potential application in telomerase-related cancer diagnosis.

PO-446 Anti-tumour efficiency of 20A, a novel G-quadruplex ligand, in in vitro and in vivocancer models: ATM and autophagy interplay

IntroductionG-quadruplexes are non-canonical DNA structures that can be stabilised by molecules called G-quadruplex ligands (G4L). G4L were first found to target telomeres and inhibit telomerase activity in cancer cells, limiting cell proliferation and making them attractive for cancer therapy. However, it has been proposed that some G4L can exert anti-tumour properties through telomerase-independent mechanisms. In this line, we investigated the underlying molecular mechanisms and anti-tumour effects of a novel G4L (20A) in several cancer models.Material and methodsWe assessed the in vitro anti-proliferative capacity of 20A in cervical carcinoma (HeLa), lung adenocarcinoma (A549), and osteosarcoma (Saos-2) cell lines by measuring cell viability, senescence and apoptosis.We used two in vivo cancer models: HeLa-cell subcutaneous and A549-cell lung orthotopic xenograft mouse models. Mice were treated by peri-tumoral and intraperitoneal 20A injections, respectively.Activation of the DNA Damage Response (DDR) and autophagy pathways were assessed by western blot analysis of the DDR kinase ATM phosphorylation and the autophagic marker LC3-II accumulation, respectively. Targeted genetic depletion of ATM and autophagic genes (ATG5 and ATG7) allowed analysing their role in 20A cytotoxicity.Results and discussionsWe found that 20A treatment causes a significant and rapid cell growth inhibition in three different types of cancer cell lines. This effect is associated with senescence induction and apoptosis through a p53-independent mechanism. Moreover, we show that 20A significantly reduces tumour growth in both in vivo cancer models used.To gain further insight into the molecular mechanisms involved in 20A cytotoxicity, we performed transcriptomic analysis which revealed functional enrichment in DDR and autophagy pathways upon 20A treatment. We confirmed these data by western blot and found that ATM is required for 20A-induced autophagy. Finally, disruption of both DDR and autophagy compromised senescence onset and increased apoptosis.ConclusionIn this study, we revealed the anti-tumour properties of this new G4L 20A in diverse in vitro and in vivo cancer models. We also show that targeting the ATM/autophagy pathway could potentiate 20A therapeutic efficiency. To our knowledge, this is the first evidence of the pivotal role of the ATM/autophagy axis in the decision between senescence and apoptosis in response to a G4L.Together, our data support further studies about 20A as a potential anti-cancer drug.

Modulation of Telomerase Activity in Cancer Cells by Dietary Compounds: A Review.

Telomerase is expressed in ~90% of human cancer cell lines and tumor specimens, whereas its enzymatic activity is not detectable in most human somatic cells, suggesting that telomerase represents a highly attractive target for selective cancer treatment. Accordingly, various classes of telomerase inhibitors have been screened and developed in recent years. We and other researchers have successfully found that some dietary compounds can modulate telomerase activity in cancer cells. Telomerase inhibitors derived from food are subdivided into two groups: one group directly blocks the enzymatic activity of telomerase (e.g., catechin and sulfoquinovosyldiacylglycerol), and the other downregulates the expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of human telomerase, via signal transduction pathways (e.g., retinoic acid and tocotrienol). In contrast, a few dietary components, including genistein and glycated lipid, induce cellular telomerase activity in several types of cancer cells, suggesting that they may be involved in tumor progression. This review summarizes the current knowledge about the effects of dietary factors on telomerase regulation in cancer cells and discusses their molecular mechanisms of action.

Molecular docking based screening of triterpenoids as potential G-quadruplex stabilizing ligands with anti-cancer activity.

Triterpenoids isolated from Ganoderma lucidum (GLTs) exhibit a broad spectrum of anti-cancer properties, including anti-proliferative, anti-metastatic and anti-angiogenic activities. Current research studies revealed the role by GLTs in inducing apoptosis and suppression of telomerase activity of cancer cells with much lower toxicity to healthy cells. Compounds selectively binding and stabilizing G-quadruplex structures could inhibit the telomerase or downregulate the oncogenes and may act as anti-cancer agents. Targeting human telomeric G-quadruplex DNA could be one of the mechanisms by which these GLTs exert anti-cancer activity. In this study, 208 GLTs were screened for ligands with high binding affinity and selectively to stabilize the pG4DNA by using the docking tool AutoDock4. The results showed that ganoderic acid A and ganoderic acid Df exhibit high binding affinity and selectively bind to the lateral groove of pG4DNA. Based on our findings, we suggest that the triterpenoid represents a new class of G-quadruplex groove binding ligands and thus act as potential anti-cancer agents.

Development of an Electrochemical Impedimetric Biosensor for the Detection of Telomerase Activity in Cancer Cells

In the last decade, a great amount of effort has gone into developing methods and devices that detect early signs of cancer diseases fast and with high efficiency. Telomerase based biosensors are an attractive alternative that comply with this requirements. Telomerase serves as a cancer biomarker because it is present in the vast majority of cancers. This enzyme elongates telomeres at the end of the chromosomal DNA, which causes cancer cells to become immortal. In this work, we developed a modified inter-digitated array gold microelectrode as the sensing platform with Electrochemical Impedance Spectroscopy (EIS) as the measuring technique for the telomerase activity. The resulting DNA biosensor detects telomerase activity in total protein extracts from Jurkat T-acute lymphoblastic leukemia cells. This simple system is a good proof of concept for a fast and efficient method that can detect telomerase activity in cancer cells.

Development of an Electrochemical Impedimetric Biosensor for the Detection of Telomerase Activity in Cancer Cells

Developing novel strategies to effectively detect biomarkers is of utmost importance in the early detection of cancer cells to increase the chances of a successful therapy. The proposed biosensor microchips works by tethering a biomolecular probe on the interdigital gold electrode array surfaces. This biomolecule probe can be customized according to the target biomarker, in order to recognize the change of conformation or bond upon contact. Telomerase is an enzyme responsible for the elongation of the telomeres between each cell division, restoring their length and avoiding cell death. This enzyme is responsible for the immortalization of cancer cells and it consists of a reverse transcriptase bound to a single stranded RNA (ssRNA) that is used as a template for telomere lengthening. The innovative research proposed in this project takes advantage of the telomerase and its ssRNA template by tethering a telomere-like ssDNA biomolecule probe that consists of a spacer composed of ten thymine, and the repeating sequence present in the telomeres of eukaryotic cells, 5’-TTAGGG-3’. The telomerase binds and elongates the probe, blocking electronic and capacitive processes on the microelectrode array surfaces. This change is detected by measuring the impedance using electrochemical impedance spectroscopy, which is correlated to the activity of telomerase and the presence of cancer cells. Moreover, telomerase is currently the target of many pharmaceutical companies to block cancer cell proliferation due to its presence in ca. 85% of known cancer cells (e.g. prostate, breast, and colorectal cancers). This project provides a unique opportunity to study the surface chemistry, impedance sensing, detection limits, and robustness of this microchip technology for its possible use at the point-of-care. This is funded by NSF Grant titled "Label-Free Electrochemical Capacitance DNA Sensing with Passive Wireless Radio Frequency Identification Sensor Technology" CHE-1152940.

Ruxolitinib therapy and telomere length in myelofibrosis

Telomeres are specialized structures of repetitive nucleotide sequences that cap the ends of human chromosomes. Their main purpose is to maintain genome stability and integrity, and to protect the cell from progressive DNA shortening during repeated division. Human enzyme telomerase complex maintains the length of telomere repeats.1 Despite the high levels of telomerase activity in cancer cells, telomere shortening can still occur. Some recent reports describe reduced telomere length in myelofibrosis (MF) regardless of hydroxycarbamide therapy, suggesting a possible prognostic relevance for this biomarker.2, 3, 4 As yet, no studies have investigated telomere dynamics following treatment with ruxolitinib, a JAK1/2 inhibitor approved for the treatment of intermediate-2 and high risk MF, primary or post-polycythemia vera (PV) and essential thrombocytemia (ET).5

Plumbagin triggers DNA damage response, telomere dysfunction and genome instability of human breast cancer cells.

Natural plant products are increasingly being used in cancer therapeutic studies due to their reduced normal cell toxicity. In this study, the anti-cancer properties of plumbagin, a naphthoquinone derivative extracted from the roots of Plumbago, were evaluated in breast cancer cells. To evaluate the effects of plumbagin on breast cancer cell types, we employed a variety of techniques comprising cell viability, cell cycle assay, comet assay, western blotting, immunocytochemistry, measurement of telomerase activity, telomere restriction fragment length, quantitative fluorescence in situ hybridisation, along with gene expression analysis of untreated cells. Plumbagin treatment induced cytotoxicity in human breast cancer cells along with cell cycle arrest, DNA damage and cell death leading to apoptosis. Plumbagin was also found to suppress the telomerase activity in cancer cells accompanied by telomere attrition. Telomere shortening was corroborated by reduced telomere fluorescence on chromosome ends and genome instability. Together, these findings may suggest the application of plumbagin as adjuvant modality in breast cancer therapeutics.

Coupling a DNA-Based Machine with Glucometer Readouts for Amplified Detection of Telomerase Activity in Cancer Cells.

The strong correlation between cancer and telomerase activity has inspired the development of new strategies to evaluate telomerase activity. Here, a personal glucose meter (PGM) system that uses DNA-based machine amplification to detect telomerase in cancer cells is reported. In this assay, telomerase elongation products are amplified in the form of another type of product by a DNA-based machine. This process can only be activated by the hybridization of the extended telomerase substrate (TS) probe and the complementary primer in the presence of telomerase. The obtained products are then transformed to glucose-related signals via a three-component assay, which enables the simple use of a PGM to indirectly quantify the telomerase activity. The proposed method realizes sensitive telomerase activity detection down to 20 HeLa cells with a significantly enhanced dynamic range. Additionally, short telomerase elongation products, such as telomerase substrate probes with two repetitive sequences, that cannot be detected using the most widely used telomeric repeat amplification protocol assay were detected.

Zero-Background Helicase-Dependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer-Dimer Artifacts.

Primer-dimer artifacts resulting from unintended template-independent primer-primer interactions often hinder the specific amplification of nucleic acids. We demonstrate, for the first time, zero-background helicase-dependent amplification (HDA), with low concentrations of both ATP and dNTPs. This strategy achieved the reliable evaluation of telomerase activity in cancer cells by eliminating primer-dimer artifacts, which have plagued many previous methods with reduced specificity. We found that the performance of the telomerase assay by zero-background HDA was negatively affected by highly concentrated cellular proteins. This inhibitory effect is attributed to the binding of DNA templates to proteins, thus making them unavailable for polymerases. However, gold nanoparticles were demonstrated to highly attenuate such inhibition by abundant proteins, and to enhance the assay sensitivity and reliability when the reaction was performed with concentrated cell extracts.

The addition of a spin column step in the telomeric repeat application protocol removes telomerase inhibitors

Telomerase activity in cancer cells is commonly analyzed by a polymerase chain reaction (PCR)-based assay termed the telomeric repeat amplification protocol (TRAP). However, nonspecific inhibition of Taq polymerase during the PCR step is frequently observed in inhibitor analysis or drug screening. Thus, the removal of excess inhibitors prior to PCR is an essential step for the proper evaluation of telomerase inhibitory effects. Here, a size exclusion spin column was applied to remove small molecular weight inhibitors from the telomerase extension products. The spin column-added protocol, termed sTRAP, provides a more reliable estimation of the inhibitory effects of telomerase activity.

Inhibition of telomerase activity by NME2: impact on metastasis suppression?

Though anti-metastatic function of non-metastatic 2 (NME2) has been implicated in multiple cancers, mechanisms of metastases control by NME2 are not clearly understood. Recent observations indicating the involvement of telomerase, the ribonucleoprotein required for telomere synthesis, in metastatic outcome are interesting. Notably, though the role of telomerase dysfunction in tumorigenesis is relatively well studied, involvement in metastasis progression is poorly understood. Recent findings demonstrate NME2 presence at telomere ends, association with telomerase, and NME2’s role in inhibition of telomerase activity in cancer cells. These present a novel opportunity to investigate mechanisms underlying NME2-mediated metastasis suppression.

HTLV-1 bZIP factor impedes the menin tumor suppressor and upregulates JunD-mediated transcription of the hTERT gene

Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by the human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD to enhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression in ATL cells confirms that these proteins are differentially involved in telomerase regulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.

Label-free highly sensitive detection of telomerase activity in cancer cell by chemiluminescence imaging

We have developed a new methodology for label-free highly sensitive telomerase activity assay using chemiluminescence imaging. This method can detect the telomerase activity from as little as 10 cultured cancer cells without PCR. Furthermore, telomerase inhibition is shown, demonstrating the potential for screening of telomerase inhibitors as anticancer drug agents.

Abstract 1234: Development of a quick sensitive real-time PCR assay for measuring telomere length

Abstract Telomeres are the ends of linear chromosomes with tandem hexameric nucleotide repeats. Its function is to protect the natural ends of chromosomes from being recognized as damaged DNA, hence contributing to chromosomal stability. Telomeric sequences shorten each time the DNA replicates; when the telomere shrinks to a certain threshold, the cell can no longer divide. Its metabolism slows down, the cell ages, and dies. This molecular clock mechanism is also associated with cancer progression and therapy. Turning off telomerase activity by shutting off telomerase activation in cancer cells will led to cellular senescence. The current gold standard method to measuring telomere length is by Southern blot; however, it is tedious and costly, and often takes up to two days to complete. Here, we describe a new, simplified automated real-time PCR assay to accurately detect the abundance of these 5’-TTAGGG-3’nucleotide repeats and determine frequency and the length of Telomere in 47 min. The assay demonstrated to be highly reproducible and accurate for human peripheral cells. The result has good correlation of R2=0.9939, slope=3.41, sensitivity to 1ng of gDNA and has an amplification efficiency of 100%. We evaluated this assay with 299 healthy human subjects to date ranging from 0 (core blood) to 99-year of age. The results are positive with an inverse correlation between telomere length and age, as telomeres were shorter with aging. The telomere length was measured in 13 samples using the gold standard Southern blot method and the new q-PCR method (R2 = 0.8623), significantly higher than the originally described (Cawthon NAS 2002). We believe this fast, sensitive methodology is a powerful tool and provides a new avenue for investigators to address the full extent and significance of telomere shortening in the tumorigenic process. This new assay described here will allow for more widespread use of this technique among cancer researchers and for study of age related telomere diseases and conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1234. doi:10.1158/1538-7445.AM2011-1234

Differential regulation of MRN (Mre11–Rad50–Nbs1) complex subunits and telomerase activity in cancer cells

Several lines of evidence suggest that cancer progression is associated with up-regulation or reactivation of telomerase and the underlying mechanism remains an active area of research. The heterotrimeric MRN complex, consisting of Mre11, Rad50 and Nbs1, which is required for the repair of double-strand breaks, plays a key role in telomere length maintenance. In this study, we show significant differences in the levels of expression of MRN complex subunits among various cancer cells and somatic cells. Notably, siRNA-mediated depletion of any of the subunits of MRN complex led to complete ablation of other subunits of the complex. Treatment of leukemia and prostate cancer cells with etoposide lead to increased expression of MRN complex subunits, with concomitant decrease in the levels of telomerase activity, compared to breast cancer cells. These studies raise the possibility of developing anti-cancer drugs targeting MRN complex subunits to sensitize a subset of cancer cells to radio- and/or chemotherapy.

Reprogramming Murine Telomerase Rapidly Inhibits the Growth of Mouse Cancer Cells In vitro and In vivo

Telomerase plays a critical role in cancer, prompting the pursuit of various telomerase-based therapeutic strategies. One such strategy, telomerase interference, exploits the high telomerase activity in cancer cells and reprograms telomerase to encode "toxic" telomeres. To date, telomerase interference has been tested in human cancer cells xenografted into mice, an approach that does not recapitulate spontaneous malignancy and offers few insights about host toxicities, because human telomerase is targeted in a mouse host. To address these limitations, we designed and validated two new gene constructs specifically targeting mouse telomerase: mutant template mouse telomerase RNA (MT-mTer) and small interfering RNA against wild-type mouse telomerase RNA (α-mTer-siRNA). Using lentiviral delivery in mouse prostate cancer cells, we achieved α-mTer-siRNA-mediated knockdown of wild-type mTer (80% depletion) and concurrent overexpression of MT-mTer (50-fold). We showed that the two constructs effectively synergize to reprogram murine telomerase to add mutant instead of wild-type telomeric repeats, resulting in rapid telomeric uncapping (5-fold increase in DNA damage foci). This, in turn, led to rapid and significant apoptosis (>90% of cells) and growth inhibition in vitro (90% reduction in viable cell mass) and in vivo (75% reduction in tumor allograft wet weight). In summary, we have shown that mouse cancer cells are vulnerable to direct telomerase interference using novel murine telomerase-targeting constructs; this approach can now be used to study the true therapeutic potential of telomerase interference in mouse spontaneous cancer models.

Telomerase interference in combination with docetaxel therapy for maximal prostate cancer inhibition: In vitro data for a novel therapeutic approach

e14596 Background: Metastatic castration-resistant prostate cancer (mCRPC) carries a median survival of 18 months with standard docetaxel based therapies. Telomerase interference (TI) is a promising novel therapeutic strategy that exploits the high telomerase activity in cancer cells by introducing a mutated telomerase RNA (MT-Ter) that encodes toxic telomeres and rapidly induces apoptosis. We investigated whether TI can be combined with docetaxel therapy to achieve greater growth inhibition in mCRPC. Methods: PC3 and DU145 mCRPC cell lines were treated with docetaxel in the presence of TI or vector control. TI was accomplished by concurrent lentiviral expression of 2 constructs: telomerase RNA with an altered template region (MT-Ter) and siRNA targeting wild-type telomerase RNA (anti-Ter siRNA). Telomere length and telomerase activity were assessed using RT-PCR and TRAP, respectively. Proliferation, apoptosis, and DNA damage were quantified using MTS, TUNEL, and 53bp1 staining, respectively. Statistical significance was calculated using a 2-sided t-test. Results: Docetaxel (10nM) induced 22% inhibition (p=0.01) of PC3 proliferation in the presence of vector control and 41% inhibition (p=0.001) in the presence of TI (results were similar in DU145 cells). This near-doubling of efficacy was attributable to an independent inhibitory effect (17% inhibition, p=0.04) from TI treatment alone, which occurred without change in bulk telomere length or telomerase activity. TI alone generated increased numbers of DNA damage foci (7/cell vs. 2/cell with vector control) while docetaxel alone did not generate significant increases in DNA damage. Both TI and docetaxel induced a marked increase in the rate of apoptosis. Conclusions: Docetaxel and TI each exerted a pro-apoptotic effect which, when combined, produced an additive inhibition of mCRPC proliferation. TI-mediated apoptosis ensued from DNA damage, consistent with its known telomeric-uncapping effect, while docetaxel-induced apoptosis was not associated with direct DNA damage, also consistent with known docetaxel mechanisms of action. These findings underscore the therapeutic promise of combining standard agents with TI to improve efficacy and reduce toxicity. No significant financial relationships to disclose.