Telomerase Activity In Cancer Cells Research Articles

TERT Promoter Methylation Is Oxygen-Sensitive and Regulates Telomerase Activity.

Telomere repeats protect linear chromosomes from degradation, and telomerase has a prominent role in their maintenance. Telomerase has telomere-independent effects on cell proliferation, DNA replication, differentiation, and tumorigenesis. TERT (telomerase reverse transcriptase enzyme), the catalytic subunit of telomerase, is required for enzyme activity. TERT promoter mutation and methylation are strongly associated with increased telomerase activation in cancer cells. TERT levels and telomerase activity are downregulated in stem cells during differentiation. The link between differentiation and telomerase can provide a valuable tool for the study of the epigenetic regulation of TERT. Oxygen levels can affect cellular behaviors including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen in driving TERT promoter modifications in embryonic stem cells (ESCs) is poorly understood. We adopted a monolayer ESC differentiation model to explore the role of physiological oxygen (physoxia) in the epigenetic regulation of telomerase and TERT. We further hypothesized that DNMTs played a role in physoxia-driven epigenetic modification. ESCs were cultured in either air or a 2% O2 environment. Physoxia culture increased the proliferation rate and stemness of the ESCs and induced a slower onset of differentiation than in ambient air. As anticipated, downregulated TERT expression correlated with reduced telomerase activity during differentiation. Consistent with the slower onset of differentiation in physoxia, the TERT expression and telomerase activity were elevated in comparison to the air-oxygen-cultured ESCs. The TERT promoter methylation levels increased during differentiation in ambient air to a greater extent than in physoxia. The chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B ChIP (Chromatin immunoprecipitation) demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B promoter binding. In conclusion, we have demonstrated that DNMT3B directly associates with TERT promoter, is associated with differentiation-linked TERT downregulation, and displays oxygen sensitivity. Taken together, these findings help identify novel aspects of telomerase regulation that may play a role in better understanding developmental regulation and potential targets for therapeutic intervention.

Application of different analysis methods to detect telomerase activity in cancer cells

Telomerase plays an important role in abnormal cell proliferation, metastasis, stem cell maintenance, and immortality in different types of cancer. As a result, the design of drugs targeting telomerase is of great significance for the treatment of cancer. In recent years, treatment strategies for telomerase have attracted extensive attention. However, finding an effective treatment is still more difficult. Understanding the structure, function and biological process of telomerase and their related proteins is of great help to drug development and clinical treatment. As a result, this research mainly introduces different biological detection methods of telomerase, including PCR, western blot, CCK-8 assay, sphere formation assay, immunocytochemistry and nanobiotic detection technology. By understanding the detection range, different aspects of detection, and advantages and disadvantages of several detection methods, the development of telomerase detection technology can be well understood. And it can better understand the activity and expression of telomerase, and hope to provide reference for future drug development targeting telomerase, telomerase and related proteins.

Telomerase gene therapy: a remission toward cancer.

Telomerase is an enzyme which is culpable for the aliment and stability of telomeres. It also maintains the genomic integrity and chromosomal stability. The progressive shortening of telomeres may cause chromosomal instability and alternation in the telomerase. It may cause telomere attrition which can lead to oncogenic incidence in human. Cancer is a disease which is induced by genetic alternations in genes. The genetic mutation within the hTERT is a common type of scenario which is generally found above 90% of cancer. In cancer, the length of telomere and the activity of telomerase are very important for cancer cells to proliferate and also for the survival of tumors. Cancer cells regulate through several pathways to increase telomerase activity. There have been several advancements developed to inhibit the telomerase activity in cancer cell but the repercussion of those have demonstrated many adverse effects. Research on AAVs-mediated telomerase gene therapy has demonstrated prominent outcomes in animal trials. Thus, it has the potential to bring significant shine in the telomerase cancer therapeutics. Here, in this review article, we have analyzed studies related to telomerase gene therapeutics to cure cancer. We also have summarized the telomerase function and mechanism of action to cause cancer. Moreover, other current development in the clinical advances of telomerase inhibition in cancer is described.

Enzymatic DNA repair cascade-driven fluorophore encoding for sensitively sensing telomerase activity in cancer cells

Telomerase is an important reverse transcriptase that is in charge of maintaining telomeres length, and the dysregulation of telomerase activity is closely associated with cancers. Accurate measurement of telomerase activity is critical to clinical diagnosis and therapeutics. Herein, by taking advantage of in vivo repair mechanisms and single-molecule detection, we demonstrate the enzymatic DNA repair cascade-driven fluorophore encoding for sensitively sensing telomerase activity in cancer cells. The presence of telomerase enables repetitive addition of (TTAGGG)n to the 3′-OH end of telomerase substrate (TS) primer to generate long telomeric products. Telomeric product then hybridizes with the apurinic (AP) probe to induce the cleavage of AP site in dsDNA by APE1, yielding a ssDNA with a biotin and an OH group at 5′ and 3′ ends and simultaneously releasing the telomeric product which in turn hybridizes with new AP probes to yield numerous ssDNAs. By adding streptavidin-coated MBs, the ssDNAs are captured and subsequently functioned as primers to initiate template-free TdT-catalyzed random incorporation of dATPs and Cy5-dATPs into 3′-OH ends, producing long poly-A chains with multiple Cy5 molecules encoding and finally forming Cy5-ssDNA-MB complexes. After magnetic separation, Cy5-ssDNA-MBs are digested by Exo I, releasing large numbers of Cy5 molecules which can be measured by single-molecule detection. This strategy exhibits excellent specificity and high sensitivity with a limit of detection (LOD) of 1 cancer cell. Moreover, it can be employed to screen inhibitors and discriminate different cancer cell lines from normal cell lines, holding enormous potential in clinical diagnosis and therapeutics.

A dual-amplification label-free ratiometric fluorescent sensor for accurate monitoring of telomerase activity based on unique intercalation characteristics of dyes toward different DNA structures

Accurate evaluation and reliable quantitation of telomerase activity in various cancer cells is of significant importance to early cancer diagnosis and therapy. All previously reported ratiometric fluorescence telomerase activity assays inevitably required the expensive labeling or sophisticated probe synthesis, which were laborious and time-consuming. To overcome such restrictions, herein we developed, for the first time, a facile and label-free ratiometric fluorescence sensing strategy for accurately and sensitively detecting telomerase activity based on unique intercalation characteristics of dyes toward double-stranded DNA and G-quadruplex, respectively. By taking advantage of the dual amplification method consisting of catalytic hairpin assembly and exonuclease III assisted cleavage reaction, telomerase activity can be quantitatively detected in MCF-7 cells ranging from 50 to 2000 with a detection limit of 16 MCF-7 cells. Furthermore, our approach could distinguish the relative telomerase activity in cancer cells from that in normal cells, as well as in different cancer cells. It was also confirmed that our strategy displayed good analytical performance for screening the inhibitors for telomerase. More importantly, compared to those labeling and/or single signal-based methods for telomerase activity detection, this assay not only effectively circumvented the expensive labeling and complex probe preparation but also avoided false-positive results, thus exhibiting the appealing features of simplicity, low cost, and high accuracy. Overall, our method might be a promising tool in telomerase activity analysis and telomerase-targeted antitumor drugs screening.

Anti-Proliferative and Anti-Telomerase Effects of Blackberry Juice and Berry- Derived Polyphenols on HepG2 Liver Cancer Cells and Normal Human Blood Mononuclear Cells.

Previous studies have provided strong evidence for the anticancer activity of berry fruits. In this study, we investigated the effects of blackberry juice and three berry- polyphenolic compounds on cell proliferation and telomerase activity in human hepatoma HepG2 and normal peripheral blood mononuclear cells (PBMCs). The cell viability and telomerase activity were measured by MTT and TRAP assay, respectively. Berry effects on the expression of genes were determined by quantitative RT-PCR assay. Blackberry, gallic acid, and resveratrol inhibited proliferation of both HepG2 and PBMC cells in a dosedependent manner. Resveratrol was more effective than gallic acid for reducing the viability of HepG2 cells, but both showed the same level of growth inhibition in PBMC cells. Berry, resveratrol, and gallic acid significantly inhibited telomerase activity in HepG2 cells. The antiproliferative effect of berry was associated with apoptotic DNA fragmentation. Gallic acid was more effective for reducing telomerase activity than resveratrol, but anthocyanin moderately increased telomerase activity in cancer cells. Telomerase activity was induced by all three polyphenols in PBMCs. Overall, Krumanin chloride was more effective to induce telomerase than gallic acid and resveratrol in PBMC cells. There was no significant difference in hTERT, hTR, and Dnmts expressions between berry treated and the control untreated HepG2 cells. But, a significant downregulation of HDAC1 and HDAC2 and upregulation of SIRT1 were observed in berry-treated cells. These data indicate that the berry anticancer effect is associated with antitelomerase activity and changes in HDACs expression. The data also suggest that berry antitelomerase activity is mainly related to its gallic acid and resveratrol, but not anthocyanin content.

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

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

Single Molecule Investigations of Protein and Small Molecule Interactions with G-Quadruplexes and their Impact on Telomere Structure

G-quadruplex (GQ) structures form in guanine-rich segments of the genome and are particularly more prominent at telomeres and promoters. Telomeric GQ structures (GQs) have emerged as potential targets for small molecule drugs that aim to inhibit telomerase activity in cancer cells, while GQs in promoters are considered to play a role in transcription and translation level gene expression regulation. Due to their high thermal stability, GQs require activity of DNA binding proteins or helicases to be unfolded. Stabilizing small molecules are typically characterized based on their impact on thermal stability of GQs. As a physiologically more relevant approach, we determined the efficacy of several prominent small molecules based on their impact of helicase activity. We measured binding kinetics of individual small molecules and their impact on dynamics and steady state Bloom helicase (BLM) mediated GQ unfolding activities. We also studied Replication Protein A (RPA), GQ, and small molecule interactions while the GQ is maintained under tension by a looped DNA. Finally, we present FRET-PAINT measurements where we studied the accessibility of long telomeric repeats that form multiple tandem GQs and observed length dependent compaction in telomere structure.

Aggregation-induced emission luminogens reveal cell cycle-dependent telomerase activity in cancer cells.

Telomerase acts as an important biomarker for tumor identification, and synthesizes telomeric repeats at the end of chromosome telomeres during the replicative phase of the cell cycle; thus, the expression level of telomerase changes as the cell cycle progresses. TERT mRNA expression and telomerase activity were significantly increased in over 80% of human cancers from tissue specimens. Although many efforts have been made in detecting the activity of TERT mRNA and active telomerase, the heterogeneous behavior of the cell cycle was overlooked, which might affect the accuracy of the detection results. Herein, the AIEgen-based biosensing systems of PyTPA-DNA and Silole-R were developed to detect the cellular level of TERT mRNA and telomerase in different cell cycles. As a result, the fluorescence signal of cancer cells gradually increased from G0/G1, G1/S to S phase. In contrast, both cancer cells arrested at G2/M phase and normal cells exhibited negligible fluorescence intensities. Compared to normal tissues, malignant tumor samples demonstrated a significant turn-on fluorescence signal. Furthermore, the transcriptomics profiling revealed that tumor biomarkers changed as the cell cycle progressed and biomarkers of CA9, TK1 and EGFR were more abundantly expressed at early S stage. In this vein, our study presented advanced biosensing tools for more accurate analysis of the cell-cycle-dependent activity of TERT mRNA and active telomerase in clinical tissue samples.

Amentoflavone as a potential telomeric G-Quadruplex DNA stabilizing agent

Telomerase enzyme plays a pivotal role in the maintenance of telomere length and thereby increases the longevity of cancer cells. The ability of telomeric DNA sequences to form quadruplex has been identified as potential drug targets for the treatment of various cancers by interfering telomerase activity. The formation of quadruplex has diverse structural conformations and is subjected to detailed DNA-Ligand interaction studies. In the present study, we investigate the possibility of stabilizing parallel quadruplex DNA sequence d [AGGG(TTAGGG)3] at the telomeric end region with the phytochemicals present in the medicinal plant Biophytum sensitivum L. Molecular docking studies reveal that the amentoflavone, a biflavonoid present in the plant binds to the quadruplex DNA and thereby stabilizing the structure. ADMET properties of amentoflavone were also investigated to understand its pharmacokinetics and pharmacological effects. Quadruplex DNA structures are seen to be stabilized in-vitro using cations K+ or Na+. This was further confirmed using molecular dynamics simulation studies of quadruplex DNA structure with K+ and Na+ along with amentoflavone. Though K+ and Na+ orient themselves in the center of the planar structure, amentoflavone binds to the quadruplex DNA structure and thereby stabilizing it. These results show that amentoflavone can stabilize the quadruplex DNA structure as good as the monovalent cations do in-vitro. Hence it is proposed that amentoflavone could be developed as a potent inhibitor of telomerase activity in cancer cells.

Enzyme-free dual-DNA walker based on catalytic hairpin assembled DNAzyme for sensing telomerase activity

Telomerase is a potential tumor biomarker. Herein, an enzyme-free and isothermal fluorescence strategy, dual-3D DNA walker based on catalytic hairpin assembled DNAzyme (CHA-DNAzyme), for ultrasensitive detection of telomerase activity is described. Upon incubation with telomerase, the telomerase primer is elongated to form telomeric repeat sequences (TR). The elongated TR stochastically moves along the predesign track by continuous strand displacement reaction to assemble the intact DNAzyme (CHA walker). Then, the activated DNAzyme autonomously moves along the other track by cleaving DNA hairpins to release fluorescent-labeled DNA fragments from AuNPs (DNAzyme walker). As a result, a large amount of fluorescence is recovered to analyze telomerase activity. The use of CHA walker and DNAzyme walker endows the sensing strategy with high sensitivity and signal transfer efficiency. The strategy exhibits a wide analytical range from 10 to 1000 HeLa cells mL−¹ with a limit of detection of 7 cells mL−¹. The strategy not only can detect telomerase activity in different tumor cells, but also quantifies telomerase activity of cancer cells from accumulated normal cells. In addition, the practicability of the strategy is validated by detecting telomerase activity in human serum.

Circulating Tumor Cells Are Associated with Recurrent Disease in Patients with Early-Stage Non-Small Cell Lung Cancer Treated with Stereotactic Body Radiotherapy.

Although stereotactic body radiotherapy (SBRT) is effective in early-stage non-small cell lung cancer (NSCLC), approximately 10%-15% of patients will fail regionally and 20%-25% distantly. We evaluate a novel circulating tumor cell (CTC) assay as a prognostic marker for increased risk of recurrence following SBRT. Ninety-two subjects (median age, 71 years) with T1a (64%), T1b (23%), or T2a (13%) stage I NSCLC treated with SBRT were prospectively enrolled. CTCs were enumerated by utilizing a GFP-expressing adenoviral probe that detects elevated telomerase activity in cancer cells. Samples were obtained before, during, and serially up to 24 months after treatment. SBRT was delivered to a median dose of 50 Gy (range, 40-60 Gy), mostly commonly in four to five fractions (92%). Thirty-eight of 92 subjects (41%) had a positive CTC test prior to SBRT. A cutoff of ≥5 CTCs/mL before treatment defined favorable (n = 78) and unfavorable (n = 14) prognostic groups. Increased risk of nodal (P = 0.04) and distant (P = 0.03) failure was observed in the unfavorable group. Within 3 months following SBRT, CTCs continued to be detected in 10 of 35 (29%) subjects. Persistent detection of CTCs was associated with increased risk of distant failure (P = 0.04) and trended toward increased regional (P = 0.08) and local failure (P = 0.16). Higher pretreatment CTCs and persistence of CTCs posttreatment is significantly associated with increased risk of recurrence outside the targeted treatment site. This suggests that CTC analysis may potentially identify patients at higher risk for regional or distant recurrences and who may benefit from either systemic therapy and/or timely locoregional salvage treatment.

Target induced framework nucleic acid nanomachine with doxorubicin-spherical nucleic acid tags for electrochemical determination of human telomerase activity

A stable and enzyme-free method is described for highly sensitive determination of telomerase activity. It is based on the use of a framework nucleic acid (FNA) nanomachine and doxorubicin-spherical nucleic acid (DSNA) tags. Upon incubation with telomerase, the primer-tetrahedron becomes elongated to form the handed swing arm. The extended swing arm autonomously moves along the predefined track consisting of entropy-tetrahedron by consecutive strand displacement under the aid of fuel-tetrahedron. As a result, many (entropy-tetrahedron)-(fuel-tetrahedron) complexes are assembled for combining the DSNA tags. This results in an amplified electrochemical signal, typically measured at around -0.63V (Ag/AgCl). The use of an enzyme-free FNA nanomachine and of DSNA tags warrants outstandingly high stability and sensitivity. The method shows a broad dynamic correlation of telomerase activity in cell extracts. The analytical range extends from 10 to 1.0 × 104 HeLa cells mL-1 with a lower detection limit of 2 cells mL-1. The differences in telomerase activity between different cancer cells can be easily evaluated. The method was further verified by quantifying telomerase activity of cancer cells in accumulated normal cells. Therefore, the sensing method has great potential for clinical application. Graphical abstractSchematic representation of the electrochemical biosensor based on target induced framework nucleic acid nanomachine with doxorubicin-spherical nucleic acids (DSNA) tags, which can be used to the determination of telomerase activity in accumulated normal cells. dNTP: Deoxynucleotide triphosphates; FT: Fuel-tetrahedron.

Toward In Vitro Epigenetic Drug Design for Thyroid Cancer: The Promise of PF-03814735, an Aurora Kinase Inhibitor.

Thyroid cancer (TC) is a very common malignancy worldwide. Chief among the innovative molecular drug targets for TC are epigenetic modifications. Increased telomerase activity in cancer cells makes telomerase a novel target for epigenetic anticancer drug innovation. Recently, telomerase reverse transcriptase (TERT) gene promoter (TERTp) mutations (C228T and C250T) were reported at high frequency in TC cell lines and tumor biopsies. In this study, three representative TC cell lines, mutant TERTp (TPC1), mutant BRAF/TERTp (KTC2), and wild-type TERTp (WRO), were screened with a drug library composed of 51 epigenetic drugs: 14 Aurora kinase inhibitors; 23 histone deacetylase inhibitors; 5 sirtuin modifiers; 3 hypoxia-inducible factor inhibitors; 2 DNA methyltransferase inhibitors; 2 histone methyltransferase inhibitors, a histone demethylase inhibitor, and a bromodomain inhibitor. Effects of the drugs on cell growth at 48 and 72 h were compared. PF-03814735, a small-molecule inhibitor of Aurora kinase A (IC50 = 0.8 nM) and B (IC50 = 5 nM), was the most potent on KTC2 cells, whereas CUDC-101, a multitarget inhibitor, was effective on both WRO and KTC2 cells. Notably, PF-03814735 was found to be the most effective epigenetic drug on cell lines harboring the C228T mutation. In conclusion, these new findings offer specific guidance on dose and time course selection to design novel therapeutic interventions against TC using PF-03814735, and specifically target cells carrying the TERTpC228T mutation. In a larger context of drug discovery science, these findings inform new strategies to forecast optimal treatment regimens for TC, particularly with Aurora kinase inhibitors and in ways guided by epigenetic drug design.

Pre-Treatment Circulating Tumor Cell Levels Correlate with Regional and Distant Failure Rates Following Stereotactic Body Radiation Therapy for Early Stage Non-Small Cell Lung Cancer

Stereotactic body radiation therapy (SBRT) has become the standard of care for patients with medically inoperable early stage non-small cell lung cancer (NSCLC) due to good local control rates and minimal morbidity. However, of patients treated with SBRT, 10-15% patients fail regionally and 25% fail distantly. Our hypothesis underlying this prospective trial was that a high burden of circulating tumor cells (CTCs) prior to treatment is a useful prognostic marker for increased risk of recurrence following SBRT. 92 subjects with stage I NSCLC treated with SBRT were prospectively enrolled on an IRB-approved protocol. CTCs were enumerated by an assay utilizing a green fluorescent protein (GFP)-expressing adenoviral probe that detects the elevated telomerase activity in cancer cells. CTC “positivity” was defined as 1.3 GFP-positive cells per mL of peripheral blood samples. Pre-treatment samples were collected at the time of treatment consent for SBRT or at simulation. RT was delivered to a median dose of 50Gy (range, 40-60 Gy) in five fractions. Patient characteristics: female (58%), Caucasian (80%), and current (20%) or former (75%) smokers; median age was 71 years. Subjects had T1a (64%), T1b (23%), or T2a (13%) NSCLC with a median tumor size of 1.7 cm (range, 0.5-5.0 cm). Kaplan-Meier survival analysis and Cox multivariate regression analysis explored the relative effect of covariates on recurrence and survival outcomes, controlling for history of previous NSCLC, tumor size, and pre-treatment PET scan SUVmax. Receiver operating characteristic curves were generated for a series of baseline CTC values between 2 and 7 to identify if there were a threshold value prognostic of disease recurrence. With a median follow-up of 24 months (range, 2-62 months), the 2-year local, regional and distant control rates were 90.0%, 86.4%, and 86.5%, respectively. 38 of 92 subjects (40%) had a positive CTC test prior to treatment. Overall, a positive CTC test when compared to a negative test was not associated with local, nodal, or distant recurrence. A pre-SBRT cutoff of ≥5 CTCs/mL demonstrated the maximal area under the cure in predicting any event of recurrence (AUC=0.64, specificity=90.4%), therein defining favorable (n=78) and unfavorable (n=14) prognostic groups by baseline CTC. Increased risk of nodal and distant failure was observed in the unfavorable versus favorable cohorts, with 2-year freedom from nodal and distant failure of 83.9% and 75.7% versus 86.7% (HR 3.92, p=0.04) and 89.2%, (HR 4.29, p=0.03). Higher pre-treatment CTC counts appears to be predictive of increased risks of regional and distant recurrences following SBRT for early stage NSCLC, thus potentially identifying the subset of patients at highest risk of regional and distant recurrences who could benefit most from intensified or adjuvant systemic therapy.

StudiIn SilicoSenyawaTurunan8-Tersubtitusi-7-Methoxy-2h-Chromen-2-OneSebagaiPenghambatEnzim Telomerase

Adjustment of telomere length by telomerase is considered as a biological marker which determine the proliferation of cancer cells. The telomerase activity in cancer cells is potential to be targeted for cancer therapies. The aim of this study was QSAR studies to determine the descriptors that affect the anticancer activity of 8-substituted-7-methoxy-2H-chromen-2-one derivatives. The descriptors were calculated by using software MOE ® 2009.10. to design new derivative compounds that have anticancer potency, to predict pharmacokinetic properties by using preADMET, and to predict tocixity by ADMET predictor TM and interaction predict to telomerase by using Autodock 4.2.6 The results showed QSAR equation was log IC 50 = -1.358 (± 1.149) - 2.957 (± 1.019) A— AM1_LUMO + 0.041 (± 0.011) A— E_vdw + 2.367 (± 0.748) A— glob + 0.487 (± 0.100) A— log S. Fourteen of the new derivative compounds were designed based on QSAR equation revealed higher activity prediction than the lead compound and performed good absorption in the intestine. Compound (33) is predicted to have low permeability in Caco-2 cells and weakly bound to plasma proteins. Compound (50) predicted weakly bound to plasma proteins as well. Toxicity prediction showed that 9 derivatives compounds were predicted to have lower nearly equal toxicity to the toxicity of the lead compound. The study of the interaction of compounds with the receptor showed that derivatives (2), (4), (40) and (41) have the highest affinity to the receptor TERT with the binding free energy is −9.60 kcal / mol, −9.39 kcal / mol, −9.20 kcal / mol and -9.08 kcal / mol. Based on QSAR study, pharmacokinetic profile, toxicity, and the study of the interaction, four compounds have the potential to be develop as anticancer with telomerase inhibition.

Cancer therapy with a CRISPR-assisted telomerase-activating gene expression system.

Cancer is caused by a series of alterations in genome and epigenome and exists in multiple complex forms, making it difficult to be prevented and/or treated. Telomerase, an enzyme responsible for the maintenance of telomere, is silent in most normal somatic cells but activated in 90% of cancer cells, making it an excellent target for cancer therapy. Therefore, various telomerase activity inhibitors have been developed to treat cancer but all failed due to side effects. Here we acted oppositely to develop a cancer gene therapy named telomerase-activating gene expression (Tage) system by utilizing the telomerase activity in cancer cells. The Tage system consisted of an effector gene expression vector that carried a 3' telomerase-recognizable stick end and an artificial transcription factor expression vector that could express dCas9-VP64 and an sgRNA targeting telomere repeat sequences. By using Cas9 as an effector gene, the Tage system effectively killed various cancer cells, including HepG2, HeLa, PANC-1, MDA-MB-453, A549, HT-29, SKOV-3, Hepa1-6, and RAW264.7, without affecting normal cells MRC-5, HL7702, and bone marrow mesenchymal stem cell (BMSC). More importantly, a four-base 3' stick end produced by the homothallic switching endonuclease in cells could be recognized by telomerase, allowing the Tage system to effectively kill cancer cells in vivo. The Tage system could effectively and safely realize its in vivo application by using adeno-associated virus (AAV) as gene vector. The virus-loaded Tage system could significantly and specifically kill cancer cells in mice by intravenous drug administration without side effects or toxicity.

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.

Tolomeres and Cancer

Telomere protects the chromosomes in normal cells, and their shortening due to cell divisions and oxidative stress induces telomere shortening causing chromosomal instability. Telomerase is an enzyme that adds TTAGG telomeric repeats at chromosomal ends. The activity of telomerase enzyme plays a significant role in initiation and progression of cancer cells. In cancer cells the telomere length is maintained by telomerase enzyme. Cancer cells survive due to the activity of telomerase enzyme due to which the length of telomere is maintained and cell evades cell death mechanisms. In cancer cells telomere shortening or dysfunctional telomeres suppress cancer progression and development due to the activation of cellular senescence pathway. In this review we summarize telomere structure, function and the role telomere plays in cancer development and progression. Hermen J. Muller and Barbara McClintock identified telomere as a structure present at the ends of the chromosomes. The word telomere is derived from the Greek word “telos” which means ends and “meres” means part. Shorter telomere length or the complete absence of telomere induces end to end fusion of the chromosomes and ultimately cause cellular senescence or cell death. James D Watson in 1970s termed end replication problem in which during DNA replication, the DNA dependant polymerase does not replicates completely at the 5’ terminal end leaving small regions of the telomere uncopied. In 1960 Leonard Hayflick and his colleagues identified that the human diploid cell can undergo limited number of cell divisions in culture. The maximum number of divisions that a cell can achieve in-vitro is known as Hayflick limit which was termed after leonard Hayflick. When the cells reaches to a limit where they can no longer divide will eventually go under biochemical and morphological changes that eventually leads to cell cycle arrest, a process known as “cellular senescence. The telomerase is an enzyme that functions to add telomere repeats to the ends of the chromosomes and was identified in 1984 by Elizbeth and her collague. The presence of telomerase enzyme activity was also identified in human cancer cell lines by Gregg in 1989. Another study conducted by Greider and associates showed the absence of telomerase enzyme in normal somatic cell. Shay and Harley in 1990s detected the presence of telomerase activity in 90 out of 101 human tumor cell samples isolated from 12 different tumor types, whereas they have found no activity in normal somatic samples (n=50) isolated from 4 different tissue types. Since then various studies on 2600 human tumor samples have shown the telomerase activity in around 90% of different tumor cells. The existence of telomerase activity in cancer cells clearly demonstrates a major role of this enzyme in cancer pathogenesis. Telomeres plays a critical role in cancer, aging, Progeria (premature aging) and various other age related disorders due to which telomere and telomerase enzyme are recently an active area of research.

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.