Use Of Telomerase Research Articles

Highlights of This Issue

Satellite RNA transcribed from tandem repeat arrays near centromeric regions in chromosomes are aberrantly and highly expressed in the early stages of pancreatic carcinogenesis. Kishikawa and colleagues generated a mouse major satellite (MajSAT) RNA-expressing mouse model and demonstrated that MajSAT RNA increases DNA damage by inhibiting stress-responded nuclear translocation of YBX1 and accelerates tumor formation in the pancreas of Kras-mutant mice, which is consistent with previous in vitro findings. This study indicates that MajSAT RNA play a role as an intrinsic mutagen in the process of pancreatic cancer development.Unrestricted replicative growth conferred by stable telomere maintenance is one of the hallmarks of cancer, and most of human cancers achieve this through the aberrant expression of telomerase. Therapeutic use of telomerase targeting agents was limited by a substantial lag phase preceding telomere shortening and the onset of cytotoxicity. This study, by Thompson and colleagues, demonstrates that telomerase facilitates the kinetics of telomere cap formation and passage through G2 phase. Transient telomerase inhibition results in a prolonged ATM-dependent DNA-damage signal that alters the cell cycle profile. This data provides the biological rationale for exploring clinical telomerase inhibition strategies that leverage telomere-length-independent mechanisms of activity.Cancer-associated adipocytes play a vital role in ovarian cancer (OC) progression. Previous studies demonstrate effective treatment of platinum-resistant OC with epigenetic therapy. However, effect of epigenetic therapy on the tumor microenvironment (TME) is not well understood. Tang and colleagues examined the effects of a DNMT inhibitor, guadecitabine, on adipocytes in the context of the tumor microenvironment. Guadecitabine treatment of adipocytes decreased OC cell migration and invasion partly through upregulation of SUSD2, a secreted tumor suppressor. These findings suggest that in addition to direct effects on cancer cells, epigenetic therapy impcats the tumor microenvironment through altered cytokine secretion to prevent OC metastasis.The nuclear receptor CAR, activated by phenobarbital, preferentially promotes hepatocellular carcinoma (HCC) development in male mice. Now growth arrest and DNA-damage-inducible 45 beta (GADD45B) is characterized as a key target of CAR in hepatocyte proliferation. CAR directly interacts and prevents GADD45B from forming a complex with MKK6 to repress the activity of the p38 MAPK tumor suppressor. Experiments with GADD45B knockout mice demonstrate that GADD45B mediates a phenobarbital-induced and male-specific hepatocyte proliferative phenotype. Thus, this study provides new mechanistic insight into phenobarbital-promoted HCC development.

Estimation of telomerase, AFP, and AFP-L3 levels in Sudanese patients with hepatocellular carcinoma and chronic liver diseases

Telomerase enzyme activity is considered a widespread and relatively selective tumor cell marker feature that can be used for detecting the presence of various neoplastic cells. The positive activity of telomerase enzyme was also recorded in chronic liver disease and is evaluated as a more appropriate biomarker for differentiating hepatocellular carcinoma (HCC) from chronic liver diseases. The current study aimed to evaluate telomerase, AFP, and AFP-L3 levels in Sudanese patients with hepatocellular carcinoma and chronic liver diseases. The study enrolled 113 patients: HCC (n = 33), cirrhosis (n = 33), and chronic hepatitis (n = 47), jointly with 50 healthy controls. Enzyme-linked immunosorbent assay (ELISA) technique was used to estimate the telomerase, AFP, and AFP-L3 levels in blood samples. The telomerase level was elevated in 70% of HCC patients, and it was significantly higher in HCC patients compared to that in healthy controls (1.38 versus 0.35 ng/ml, respectively; P < 0.01). Serum alpha-fetoprotein (AFP) and Lens culinaris agglutinin-reactive (AFP-L3) levels were also significantly elevated in HCC patients compared to healthy controls (130.76 versus 5.59 ng/ml; P < 0.01) and (59.76 versus 0.49 ng/ml; P < 0.01), respectively. The combination between telomerase and AFP or AFPL-3 lead to increased telomerase sensitivity (85 and 79%, respectively).Telomerase was found to be further sensitive in patients with a tumor size of less than 5 cm as well as to tumors of more than 5 cm. Two healthy control samples showed an increase in telomerase level. The combined use of telomerase, AFP, and AFP-L3 levels might be useful for achieving better sensitivity for HCC patients.

NMR assignments of the N-terminal domain of Ogataea polymorpha telomerase reverse transcriptase.

Telomerase is a ribonucleoprotein enzyme that adds telomeric DNA fragments to the ends of chromosomes. This enzyme is the focus of substantial attention, both because its structure and mechanism of action are still poorly studied, and because of its pivotal roles in aging and cellular proliferation. The use of telomerase as a potential target for the design of new anticancer drugs is also of great interest. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is essential for activity and processivity. Elucidation of the structure and dynamics of TEN in solution is important for understanding the molecular mechanism of telomerase activity and for the design of new telomerase inhibitors. To approach this problem, in this study we report the (1)H, (13)C, and (15)N chemical shift assignments of TEN from Ogataea polymorpha. Analysis of the assigned chemical shifts allowed us to identify secondary structures and protein regions potentially involved in interaction with other participants of the telomerase catalytic cycle.

Telomerase and hTERT: Can they serve as markers for gastric cancer diagnosis?

To investigate telomerase activity and human telomerase reverse transcriptase (hTERT) expression in normal human gastric mucosal epithelial cells (nhGMECs) and fibroblasts (nhGMFs). nhGMECs and nhGMFs were isolated and cultured from specimens obtained during routine surgery for bleeding peptic ulcer. Telomerase activity in nhGMFs, nhGMECs, and the tumor cell lines BGC-823, SGC-7901 and MKN-28 cells was analyzed using the telomeric repeat amplification protocol assay. hTERT protein was determined in nhGMECs, nhGMFs, BGC-823, SGC-7901 and MKN-28 cells by indirect immunofluorescence. A similar level of telomerase activity was observed in nhGMECs, nhGMFs and BGC-823, SGC-7901, MKN-28 cell lines. Positive hTERT immunostaining was detected in nhGMECs, nhGMFs, BGC-823, SGC-7901 and MKN-28 cell lines. The use of telomerase or hTERT as diagnostic markers for gastric cancer may require further studies.

Znaczenie telomerazy w diagnostyce i terapii onkologicznej u kobiet po menopauzie

Telomeres and telomerase are responsible for protection and maintenance of genomic stability of all eukaryotic cells. Several studies showed that one of the most distinguishing features of cancer cells is an increased expression and activity of telomerase, in contrast to somatic cells. This gives an opportunity for use of telomerase as a specific marker of cancer lesions or even as a molecular target. However, there are some questions concerning diagnostic specificity of this marker especially in the context of high proliferation potential of normal cells, such as the uterus and ovaries. The proliferation potential, however, decreases due to the menopause syndrome. Women’s age and hormonal changes influence the functioning of many tissues, including alterations in telomerase activity of endometrial and ovarian cancer cells. The different characteristics of telomerase activity and telomere length in cells of postmenopausal women give an opportunity to utilize the telomeres and telomerase for the development of new cancer diagnostic tests and therapeutic methods. This work is aimed to summarize the information about the significance of telomeres and telomerase in the diagnosis and treatment of cancer in postmenopausal women.

Telomere maintenance and telomerase activity are differentially regulated in asexual and sexual worms

In most sexually reproducing animals, replication and maintenance of telomeres occurs in the germ line and during early development in embryogenesis through the use of telomerase. Somatic cells generally do not maintain telomere sequences, and these cells become senescent in adults as telomeres shorten to a critical length. Some animals reproduce clonally and must therefore require adult somatic mechanisms for maintaining their chromosome ends. Here we study the telomere biology of planarian flatworms with apparently limitless regenerative capacity fueled by a population of highly proliferative adult stem cells. We show that somatic telomere maintenance is different in asexual and sexual animals. Asexual animals maintain telomere length somatically during reproduction by fission or when regeneration is induced by amputation, whereas sexual animals only achieve telomere elongation through sexual reproduction. We demonstrate that this difference is reflected in the expression and alternate splicing of the protein subunit of the telomerase enzyme. Asexual adult planarian stem cells appear to maintain telomere length over evolutionary timescales without passage through a germ-line stage. The adaptations we observe demonstrate indefinite somatic telomerase activity in proliferating stem cells during regeneration or reproduction by fission, and establish planarians as a pertinent model for studying telomere structure, function, and maintenance.

Amelogenin as a target for real time PCR quantitation of forensic templates

PCR is the ubiquitous method of forensic DNA analysis, but prior to amplification, two other processes are crucial to obtaining a satisfactory result: template DNA extraction and template quantitation. Here we will presume the extraction process has been performed, and concentrate on the quantitation step: a focus of recent advances. Real time quantitative PCR (RT-QPCR) is an advantageous alternative to probe hybridisation or fluorescent dye association, which are (respectively) laborious and less accurate procedures. We reviewed the available commercial methods of RT-QPCR and concluded that for our requirements, a more attractive solution was the in-house development and validation of an ultra-rapid, small batch size solution. Our solution is real time detection using the Roche LightCycler 2.0 and the amplification of a 106/112 bp amelogenin amplicon. Melt-curve analysis and back extrapolation to the starting template-dependant crossing point generates results from 32 samples in ∼30 min (post PCR assembly) and this approach has advantages in that a positive quantitation result implies that in the SGM Plus™ amplification that follows, at the very least, an amelogenin product should be generated. The use of the amelogenin target also provides an indication of the possibility of PCR product travelling from the separate PCR product room backwards into the clean PCR set-up environment, something that the use of telomerase or β-globin amplicons cannot provide. We have validated the use of our LightCycler-amelogenin based quantitation system and have seen significant improvements in the reliability of quantitation measurements in our forensic laboratories.

Tissue engineered vessels: Cells to telomeres

Vascular diseases continue to affect people of all ages, and therapies are continually being improved. Cardiovascular tissue engineering is becoming an established field that will enable the replacement or modification of diseased tissues. Although commonly thought to affect older populations, cardiac disease afflicts many pediatric patients who come with a different set of needs. Particularly, with congenital defects, an engineered construct must be able to grow with the patient to avoid costly, repeated surgeries. Of the wide variety of congenital cardiac defects observed today, many concern the vessels in and around the heart. The selection of a proper cell source and attention to cellular lifespan are important factors that dictate the success of a vascular graft. Engineered blood vessels have been constructed from both synthetic and biological materials, using cells from human, bovine, and porcine species. Smooth muscle and endothelial cells taken from vessel explants, as well as cells derived from bone marrow, have been utilized to generate extracellular matrix components needed for conduit construction. The limited lifespan of these cells, especially after weeks in culture, has been addressed with the use of the telomerase enzyme. The catalytic subunit of telomerase, hTERT, has been used to maintain telomere length and consequently extend cellular lifespan. This review looks at current research that is expanding the vessel tissue engineering field by implementing novel use of cells and telomerase.

Use of Telomerase to Create Bioengineered Tissues

Telomeres are repetitive DNA (TTAGGG) elements at the ends of chromosomes. Telomerase is a ribonucleoprotein complex that catalyzes the addition of telomeric sequences to the ends of chromosomes. The catalytic protein component of telomerase (hTERT) is expressed only in specific germ line cells, proliferative stem cells of renewal tissues, and cancer cells. The expression of hTERT in normal cells reconstitutes telomerase activity and circumvents the induction of senescence. Telomeres shorten with each cell division, eventually leading to senescence (aging), due to incomplete lagging DNA strand synthesis and end-processing events, and because telomerase activity is not detected in most somatic tissues. There are specific tissues and locations in which replicative senescence likely contributes to the decline in human physiological function with increased age and with chronic illnesses. While expressing hTERT in cells results in the maintenance of telomere length and greatly extended life span, blocking replicative aging systemically would be predicted to increase the potential for tumor formation. However, there are many situations in which the transient rejuvenation of cells could be beneficial. Ectopic expression of hTERT has been shown to immortalize human skin keratinocytes, dermal fibroblasts, muscle satellite (stem), and vascular endothelial, myometrial, retinal-pigmented, and breast epithelial cells. In addition, human bronchial, corneal and skin cells expressing hTERT can be used to form organotypic (3D) cultures (bioengineered tissues) that express differentiation-specific proteins, demonstrating that hTERT by itself does not alter normal physiology. The production of hTERT-engineered tissues offers the possibility of producing tissues to treat a variety of chronic diseases and age-related medical conditions that are due to telomere-based replicative senescence.

Real-time telomerase activity measurements for detection of cancer

Since the hallmark report of the PCR-based telomeric repeat amplification protocol (TRAP) in 1994, there has been a flurry of investigations of telomerase activity on normal, benign, premalignant and cancerous samples representative of the various stages of tumorigenesis. Basic research and technological advances in human genetics, biochemistry and model systems have brought much progress towards the understanding of human infectious, hereditary and somatically acquired diseases. The knowledge of carcinogenesis has increased very rapidly in the past few years, particularly with the development of automated molecular biologic analysis of tumors and preneoplastic lesions. Despite the wide variety of studies on the potential use of telomerase as a cancer biomarker, the variability of reported telomerase activity and the lack of a transferable detection method have prevented it from becoming a routine clinical application. Real-time PCR is a clinically transferable method and the advancement of real-time measurements of telomerase will facilitate moving telomerase activity and technologies towards clinical validation. It is expected that the next 5 years will see telomerase integrated into the initial detection and follow-up monitoring of cancer patients. The hope is that the use of telomerase will finally translate into a diagnostic to help realize longer survival and a better quality of life.

The Simultaneous Use of Telomerase, Cytokeratin 20 and CD4 for Bladder Cancer Detection in Urine

Objective: Because of the low sensitivity of urinary cytological diagnosis of urinary bladder carcinoma, new molecular diagnostic methods have been proposed. We decided to verify the expression of telomerase mRNA coding for the catalytic component (hTRT), cytokeratin 20 (CK20) and CD4 antigen mRNAs in urine as possible diagnostic tool. Methods: Evaluation of hTRT, CK20, CD4 mRNAs was performed in 50 ml of naturally voided urine of 205 patients of which 153 with bladder cancer (Tis, n = 11; TaGx, n = 4; TaG1, n = 25; TaG2, n = 26; TaG3, n = 8; T1G1, n = 16; T1G2, n = 17; T1G3, n = 20; T2G2, n = 6; T2G3, n = 13; T3G3, n = 7) and 52 controls. A quantitative expression of hTRT at mRNA level versus TRAP (telomeric repeat amplification protocol) assay was performed in 20 patients and 14 controls. The expression of RT-PCR for hTRT, CK20, CD4 versus urinary cytology was analysed in 44 patients with bladder cancer. Evaluating the three molecular markers together, the result was considered correct when at least two of the markers were positive, suspected when only one marker was positive and negative for diagnosis of tumour when all markers were negative. The performance of the diagnostic model resulted from the logistic analysis evaluated with receiver operating characteristics (ROC) curve analysis. Results: The sensitivity detected for each tumour marker was as follows: for hTRT 90.8%, for CK20 84.3% and for CD4 was 64.7%, while the specificity was 94.2% for CD4 and 78.8% for both hTRT and CK20. When a simultaneous evaluation of the three tumour markers was considered, 88.2% of the diagnoses were correct, 11.8% were suspected for tumour and none were mistaken. When compared with cytology, the simultaneous use of the three markers allowed reaching a correct diagnosis in 88% of the cases in comparison to 25% by urinary cytology. The sensitivity in the detection of bladder cancer was higher for hTRT at mRNA level in comparison with the enzymatic activity detection with TRAP (90% vs. 35%) while the specificity for both markers resulted very high (100%). Conclusions: These data show that in the future the diagnostic improvement of urine based molecular markers for the detection of bladder cancer in the urine could improve the sensitivity of urinary cytology reducing the need of a cystoscopy.

Telomerase as drug and drug target for the treatment of thyroid cancer.

Telomerase is known to be activated in almost all cancer cells and is quiescent in almost all normal cells. Therefore, it follows that therapeutic strategies directed against cancer would include the targeting of telomerase, as well as the use of telomerase. Several approaches have been used both in vitro and in vivo and include the following: 1) antisense; 2) immunotherapy directed against hTERT; and 3) the use of telomerase promoter to direct cytotoxic therapy. Herein we review these approaches and discuss their potential applicability against thyroid cancer.

Telomeres and telomerase: a modern fountain of youth?

Since ageing is a universal human feature, it is not surprising that, from the Babylonian epic of Gilgamesh to Ponce de Leon seeking the "Fountain of Youth," countless people have dreamed of finding a way to avoid ageing, to no avail. Yet the search continues. In this review, we present one of the latest candidates: the enzyme telomerase, capable of elongating the tips of chromosomes, the telomeres. Research into the causes of cellular ageing established the telomeres as the molecular clock that counts the number of times cells divide and triggers cellular senescence. Herein, we review arguments both in favor and against the use of telomerase as an anti-ageing therapy. The importance of the telomeres in cellular ageing, the low or non-existent levels of telomerase activity in human tissues, and the ability of telomerase to immortalize human cells suggest that telomerase can be used as an anti-ageing therapy. On the other hand, recent experiments in mice have raised doubts whether telomerase affects organismal ageing. Results from human cells expressing telomerase have also suggested telomerase may promote tumorigenesis. We conclude that, though telomerase may be used in regenerative medicine and to treat specific diseases, it is unlikely to become a source of anti-ageing therapies.

In situ analysis of telomerase RNA gene expression as a marker for tumor progression.

AbstractLung cancer is common in men and women, has a very poor prognosis, and is therefore a major cause of premature mortality. As such, any prospects for improved therapy are of great significance (1–4). The promise of telomerase as a therapeutic target is now close to realisation with extremely encouraging preclinical studies aimed at the RNA component (hTERC) of telomerase (5–11). The rational integration of telomerase therapeutics into clinical trials will therefore require tumors to be well characterized for hTERC expression (12–14). The regulation of telomerase activity is likely to be a complex issue including the transcriptional activity of the telomerase RNA component gene hTERC and the telomerase catalytic component gene (hTERT), and the interaction of telomerase with other telomere associated proteins (10,15–20; see Fig. 1). The use of telomerase as a diagnostic marker and target for cancer therapy relies on the development of reliable assays and technologies to detect telomeres and telomerase expression (14,21–26; see Table 1 ). Molecular techniques can be roughly broken down into two groups, lysate analysis and in situ analysis (14,23,27). With lysate methods, tumor biopsies are homogenized and the spatial relationships between tumor cells are destroyed (Southern-blot analysis and polymerase chain reaction [PCR]). This leads to a loss of information on heterogeneity and small subpopulations and presents an averaging of changes. Regulation of telomerase activity. Table 1 Methods for the Analysis of Telomeres and telomerase Full size table KeywordsAcetic AnhydrideColumn BufferTelomere Associate ProteinGene hTERCRadioactive NucleotideThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Recent developments on human cell lines for the bioartificial liver.

Most bioartificial liver (BAL) devices contain porcine primary hepatocytes as their biological component. However, alternatives are needed due to xenotransplantation associated risks. Human liver cell lines have excellent growth characteristics and are therefore candidates for application in BAL devices. Tumour-derived cell lines HepG2 and C3A express a variety of liver functions, but some specific liver functions, like ammonia detoxification and ureagenesis are insufficient. Immortalised human hepatocytes might offer better prospects. The balance between immortalisation and transformation with dedifferentiation of cells seems controllable by conditional immortalisation and/or the use of telomerase as immortalising agent. Another promising approach will be the use of embryonic or adult human stem cells. Rodent stem cells have been directed to hepatic differentiation in vitro, which might be applicable to human stem cells. However, both functionality and safety of immortalised human liver cell lines and differentiated stem cells should be improved before successful use in BAL devices becomes reality.

The Generation and Characterization of a Cell Line Derived from a Sporadic Renal Angiomyolipoma: Use of Telomerase to Obtain Stable Populations of Cells from Benign Neoplasms

Angiomyolipomas are benign tumors of the kidney derived from putative perivascular epithelioid cells, that may undergo differentiation into cells with features of melanocytes, smooth muscle, and fat. To gain further insight into angiomyolipomas, we have generated the first human angiomyolipoma cell line by sequential introduction of SV40 large T antigen and human telomerase into human angiomyolipoma cells. These cells show phenotypic characteristics of angiomyolipomas, namely differentiation markers of smooth muscle (smooth muscle actin), adipose tissue (peroxisome proliferator-activator receptor gamma, PPARgamma), and melanocytes (microophthalmia, MITF), thus demonstrating that a single cell type can exhibit all of these phenotypes. These cells should serve as a valuable tool to elucidate signal transduction pathways underlying renal angiomyolipomas.

Telomerase and breast cancer.

Current therapies for breast cancer include treatments that are toxic and often result in drug resistance. Telomerase, a cellular reverse transcriptase that maintains the ends of chromosomes (telomeres), is activated in the vast majority of breast cancers (over 90% of breast carcinomas) but not in normal adjacent tissues. Telomerase is thus an attractive target for both diagnosis and therapy because of its distinct pattern of expression. We address the use of telomerase in the diagnostics of breast pathology, as well as the use of telomerase inhibitors in the treatment and prevention of breast cancer.

Increased epidermal tumors and increased skin wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes.

Telomerase transgenics are an important tool to assess the role of telomerase in cancer, as well as to evaluate the potential use of telomerase for gene therapy of age-associated diseases. Here, we have targeted the expression of the catalytic component of mouse telomerase, mTERT, to basal keratinocytes using the bovine keratin 5 promoter. These telomerase-transgenic mice are viable and show histologically normal stratified epithelia with high levels of telomerase activity and normal telomere length. Interestingly, the epidermis of these mice is highly responsive to the mitogenic effects of phorbol esters, and it is more susceptible than that of wild-type littermates to the development skin tumors upon chemical carcinogenesis. The epidermis of telomerase-transgenic mice also shows an increased wound-healing rate compared with wild-type littermates. These results suggest that, contrary to the general assumption, telomerase actively promotes proliferation in cells that have sufficiently long telomeres and unravel potential risks of gene therapy for age-associated diseases based on telomerase upregulation.

Telomerase and cancer.

Telomerase, a eukaryotic ribonucleoprotein (RNP) complex, contains both an essential RNA and a protein reverse transcriptase subunit. By reverse transcription, the telomerase RNP maintains telomere length stability in almost all cancer cells. Over the past few years there has been significant progress in identifying the components of the telomerase holoenzyme complex and the proteins that associate with telomeres, in order to elucidate mechanisms of telomere length regulation. This review covers recent advances in the field including the use of telomerase in cancer diagnostics and an overview of anti-telomerase cancer therapeutic approaches.

DNA G-quadruplexes, telomere-specific proteins and telomere-associated enzymes as potential targets for new anticancer drugs.

Telomeres and telomerase have been subjects to a tremendous attention from scientists and oncologists during the past 5 years. This interest has been motivated by the potential of telomerase as a tumor marker for the diagnosis and the prognosis of cancer. The possible use of telomerase or telomeres as new targets for anticancer drugs also triggered investigations. The expression of telomerase was found in overall 85% of cancers. Telomerase is early expressed during oncogenesis with a gradient indicating that a high level of telomerase expression could be associated with a bad prognosis. Therefore, drugs targeting telomerase and telomeres might be useful in many human tumors with little restrictions regarding the tumor type or on the stage of the disease. Moreover, since telomerase is not or slightly expressed in normal cells, it has been postulated that drugs targeting telomerase would induce low toxicity. The race for the discovery of telomerase inhibitors has started while the identification of the components controlling telomerase, telomeres, cell survival, senescence, and apoptosis was still in progress. The recent identification of components regulating telomere length and telomerase expression (TRF1, TRF2, and tankyrase) opened a variety of new opportunities to control telomerase/telomere interactions. Meanwhile, a proof of principle was provided that changing telomere interactions with telomere binding proteins by chemical or biological means can induce cancer cell death. Interestingly, recent data challenge the old paradigm which suggested that a long exposure to telomerase and telomere inhibitors is necessary to induce anticancer effects. In this paper, we review the most recent information concerning the regulation of telomere length and telomerase expression, with emphasis on mechanisms that might translate into new drug discovery.