Telomerase activity and telomere length in primary and metastatic tumors from pediatric bone cancer patients

The presence of telomerase activity has been analyzed in almost all tumor types and tumor-derived cell lines. However, there are very few studies that focus on the presence of telomerase activity in bone tumors, and most of them report analysis on very few samples or bone-derived cell lines. The objective of this study was to analyze the telomere length and telomerase activity in primary tumors and metastatic lesions from pediatric osteosarcoma and Ewing's sarcoma patients. The presence of telomerase activity was analyzed by the telomeric repeat amplification protocol assay, and the telomere length was measured by Southern blot. Results were related to survival and clinical outcome. Telomerase activity was detected in 85% of the bone tumor metastases (100% Ewing's sarcomas and 75% osteosarcomas) but only in 12% of the primary tumors (11.1% osteosarcomas and 12.5% Ewing's sarcomas). Bone tumor tissues with telomerase activity had mean telomere lengths 3 kb shorter than those with no detectable telomerase activity (p = 0.041). The presence of telomerase activity was associated with survival (p = 0.009), and longer event-free survival periods were found in patients who lacked telomerase activity compared with those who had detectable telomerase activity levels in their tumor tissues (p = 0.037). The presence of longer telomeres in primary pediatric bone tumors than in metastases could be indicative of alternative mechanisms of lengthening of telomeres for their telomere maintenance rather than telomerase activity. Nevertheless, the activation of telomerase seems to be a crucial step in the malignant progression and acquisition of invasive capability of bone tumors

Ancestral Mutation in Telomerase Causes Defects in Repeat Addition Processivity and Manifests As Familial Pulmonary Fibrosis

The telomerase reverse transcriptase synthesizes new telomeres onto chromosome ends by copying from a short template within its integral RNA component. During telomere synthesis, telomerase adds multiple short DNA repeats successively, a property known as repeat addition processivity. However, the consequences of defects in processivity on telomere length maintenance are not fully known. Germline mutations in telomerase cause haploinsufficiency in syndromes of telomere shortening, which most commonly manifest in the age-related disease idiopathic pulmonary fibrosis. We identified two pulmonary fibrosis families that share two non-synonymous substitutions in the catalytic domain of the telomerase reverse transcriptase gene hTERT: V791I and V867M. The two variants fell on the same hTERT allele and were associated with telomere shortening. Genealogy suggested that the pedigrees shared a single ancestor from the nineteenth century, and genetic studies confirmed the two families had a common founder. Functional studies indicated that, although the double mutant did not dramatically affect first repeat addition, hTERT V791I-V867M showed severe defects in telomere repeat addition processivity in vitro. Our data identify an ancestral mutation in telomerase with a novel loss-of-function mechanism. They indicate that telomere repeat addition processivity is a critical determinant of telomere length and telomere-mediated disease

Telomerase promoter mutations in cancer: an emerging molecular biomarker?

João Vinagre, Vasco Pinto and Ricardo Celestino contributed equally to the manuscript.Cell immortalization has been considered for a long time as a classic hallmark of cancer cells. Besides telomerase reactivation, such immortalization could be due to telomere maintenance through the “alternative mechanism of telomere lengthening” (ALT) but the mechanisms underlying both forms of reactivation remained elusive. Mutations in the coding region of telomerase gene are very rare in the cancer setting, despite being associated with some degenerative diseases. Recently, mutations in telomerase (TERT) gene promoter were found in sporadic and familial melanoma and subsequently in several cancer models, notably in gliomas, thyroid cancer and bladder cancer. The importance of these findings has been reinforced by the association of TERT mutations in some cancer types with tumour aggressiveness and patient survival. In the first part of this review, we summarize the data on the biology of telomeres and telomerase, available methodological approaches and non-neoplastic diseases associated with telomere dysfunction. In the second part, we review the information on telomerase expression and genetic alterations in the most relevant types of cancer (skin, thyroid, bladder and central nervous system) on record, and discuss the value of telomerase as a new biomarker with impact on the prognosis and survival of the patients and as a putative therapeutic target

Mechanisms of human telomerase reverse transcriptase (hTERT) regulation: clinical impacts in cancer

Background Limitless self-renewal is one of the hallmarks of cancer and is attained by telomere maintenance, essentially through telomerase (hTERT) activation. Transcriptional regulation of hTERT is believed to play a major role in telomerase activation in human cancers. Main body The dominant interest in telomerase results from its role in cancer. The role of telomeres and telomere maintenance mechanisms is well established as a major driving force in generating chromosomal and genomic instability. Cancer cells have acquired the ability to overcome their fate of senescence via telomere length maintenance mechanisms, mainly by telomerase activation. hTERT expression is up-regulated in tumors via multiple genetic and epigenetic mechanisms including hTERT amplifications, hTERT structural variants, hTERT promoter mutations and epigenetic modifications through hTERT promoter methylation. Genetic (hTERT promoter mutations) and epigenetic (hTERT promoter methylation and miRNAs) events were shown to have clinical implications in cancers that depend on hTERT activation. Knowing that telomeres are crucial for cellular self-renewal, the mechanisms responsible for telomere maintenance have a crucial role in cancer diseases and might be important oncological biomarkers. Thus, rather than quantifying TERT expression and its correlation with telomerase activation, the discovery and the assessment of the mechanisms responsible for TERT upregulation offers important information that may be used for diagnosis, prognosis, and treatment monitoring in oncology. Furthermore, a better understanding of these mechanisms may promote their translation into effective targeted cancer therapies. Conclusion Herein, we reviewed the underlying mechanisms of hTERT regulation, their role in oncogenesis, and the potential clinical applications in telomerase-dependent cancers.info:eu-repo/semantics/publishedVersio

Cell Proliferation in the Presence of Telomerase

BACKGROUND: Telomerase, which is active early in development and later in stem and germline cells, is also active in the majority of human cancers. One of the known functions of telomerase is to extend the ends of linear chromosomes, countering their gradual shortening at each cell division due to the end replication problem and postreplication processing. Telomerase concentration levels vary between different cell types as well as between different tumors. In addition variable telomerase concentrations will exist in different cells in the same tumor when telomerase inhibitors are used, because of limitations of drug delivery in tissue. Telomerase extends short telomeres more frequently than long telomeres and the relation between the extension frequency and the telomere length is nonlinear. METHODOLOGY/PRINCIPAL FINDINGS: Here, the biological data of the nonlinear telomerase-telomere dynamics is incorporated in a mathematical theory to relate the proliferative potential of a cell to the telomerase concentration in that cell. The main result of the paper is that the proliferative capacity of a cell grows exponentially with the telomerase concentration. CONCLUSIONS/SIGNIFICANCE: The theory presented here suggests that long term telomerase inhibition in every cancer progenitor or cancer stem cell is needed for successful telomere targeted cancer treatment. This theory also can be used to plan and assess the results of clinical trials targeting telomerase

The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase

BACKGROUND: The activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT), which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT). METHODS: Telomerase activity (TA) was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients. RESULTS: High levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome) showed no telomerase activity and only minimal hTERT expression. CONCLUSIONS: These findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length

Aneuploidy as a mechanism of adaptation to telomerase insufficiency

Cells’ survival is determined by their ability to adapt to constantly changing environment. Adaptation responses involve global changes in transcription, translation, and posttranslational modifications of proteins. In recent years, karyotype changes in adapting populations of single cell organisms have been reported in a number of studies. More recently, we have described aneuploidy as an adaptation mechanism used by populations of budding yeast Saccharomyces cerevisiae to survive telomerase insufficiency induced by elevated growth temperature. Genetic evidence suggests that telomerase insufficiency is caused by decreased levels of the telomerase catalytic subunit Est2. Here, we present experiments arguing that the underlying cause of this phenomenon may be within the telomerase RNA TLC1: changes in the expression of TLC1 as well as mutations in the TLC1 template region affect telomere length equilibrium and the temperature threshold for the induction of telomerase insufficiency. We discuss what lies at the root of telomerase insufficiency, how cell populations overcome it through aneuploidy and whether reversible aneuploidy could be an adaptation mechanism for a variety of environmental stresses