Quantitative relationship between functionally active telomerase and major telomerase components (hTERT and hTR) in acute leukaemia cells.
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ABSTRACT:
SUBMITTER: Ohyashiki JH
PROVIDER: S-EPMC2361762 | biostudies-other | 2005 May
REPOSITORIES: biostudies-other
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Project description:Telomerase is a reverse transcriptase that adds telomeric repeats to chromosomal ends. In most normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to limited proliferative life-span. Telomerase reactivation is associated with cellular immortalization and is a frequent event during tumorigenesis. The telomerase ribonucleoprotein complex consists of two essential components, a catalytic protein subunit [human telomerase reverse transcriptase (hTERT)] and a template RNA (hTR). hTR is constitutively expressed, while hTERT is almost universally absent in telomerase-negative cells. Although repression of telomerase is transcriptional in telomerase-negative cells, post-transcriptional and assembly processes are likely to play important roles in regulating telomerase activity in those that are telomerase-positive. The telomerase transcript can also be alternatively spliced into a variety of non-functional forms. To establish the quantitative relationships between telomerase activity and its various components, we determined the numbers of molecules of hTR and hTERT mRNA, and the levels of alternatively spliced hTERT mRNA variants in normal, in vitro immortalized and cancer cell lines. We report here that there is surprisingly little variation in the proportion of alternatively spliced forms of hTERT in different cell lines. The only variation observed occurred when a change in splicing to non-functional forms appeared in response to conditions that repress telomerase activity in IDH4 cells. We also found that most telomerase-positive cell lines only contain a few molecules of potentially functional hTERT mRNA, and there is a correlation between telomerase activity and the levels of both hTR and hTERT +alpha+beta mRNA.
| S-EPMC96692 | biostudies-literature
Project description:Telomerase is the ribonucleoprotein (RNP) enzyme that elongates telomeric DNA to compensate for the attrition occurring during each cycle of DNA replication. Knowing the levels of telomerase in continuously dividing cells is important for understanding how much telomerase is required for cell immortality. In this study, we measured the endogenous levels of the human telomerase RNP and its two key components, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT). We estimate ? 240 telomerase monomers per cell for HEK 293T and HeLa, a number similar to that of telomeres in late S phase. The subunits were in excess of RNPs (e.g. ? 1150 hTR and ? 500 hTERT molecules per HeLa cell), suggesting the existence of unassembled components. This hypothesis was tested by overexpressing individual subunits, which increased total telomerase activity as measured by the direct enzyme assay. Thus, there are subpopulations of both hTR and hTERT not assembled into telomerase but capable of being recruited. We also determined the specific activity of endogenous telomerase and of overexpressed super-telomerase both to be ? 60 nt incorporated per telomerase per minute, with Km(dGTP) ? 17 ?M, indicating super-telomerase is as catalytically active as endogenous telomerase and is thus a good model for biochemical studies.
| S-EPMC4117779 | biostudies-literature
Project description:Telomerase catalyzes telomeric DNA synthesis, an essential process to maintain the length of telomere for continuous cell proliferation and genomic stability. Telomerase is activated in gametes, stem cells, and most tumor cells, and its activity is tightly controlled by a catalytic human telomerase reverse transcriptase (hTERT) subunit and a collection of associated proteins. In the present work, normal human testis tissue was used for the first time to identify proteins involved in the telomerase regulation under normal physiological conditions. Immunoprecipitation was performed using total protein lysates from the normal testis tissue and the proteins of interest were identified by microfluidic high-performance liquid chromatography and tandem mass spectrometry (HPLC-Chip-MS/MS). The regulatory role of PCDH10 in telomerase activity was confirmed by a telomeric repeat amplification protocol (TRAP) assay, and the biological functions of it were characterized by in vitro proliferation, migration, and invasion assays. A new in vivo hTERT interacting protein, protocadherin 10 (PCDH10), was identified. Overexpression of PCDH10 in pancreatic cancer cells impaired telomere elongation by inhibiting telomerase activity while having no obvious effect on hTERT expression at mRNA and protein levels. As a result of this critical function in telomerase regulation, PCDH10 was found to inhibit cell proliferation, migration, and invasion, suggesting a tumor suppressive role of this protein. Our data suggested that PCDH10 played a critical role in cancer cell growth, by negatively regulating telomerase activity, implicating a potential value in future therapeutic development against cancer.
| S-EPMC5058908 | biostudies-literature
Project description:hTERT, a catalytic component of human telomerase, is undetectable in normal somatic cells but up-regulated in cancer and stem cells where telomere length is maintained by telomerase. Accumulated evidence indicates that hTERT may have noncanonical functions beyond telomerase by regulating the expression of particular genes. However, comprehensive identification of the genes regulated by hTERT is unavailable. In this report, we expressed WT hTERT and hTERTmut which displays dysfunctional catalytic activity, in human U2OS cancer cells and VA-13 immortalized fibroblast cells, both of which lack endogenous hTERT and hTR expression. Changes in gene expression induced by hTERT and hTERT-mut expression were determined by genome-wide RNA-seq and verified by qPCR. Our results showed that hTERT affects different genes in two cell lines, implying that the regulation of gene expression by hTERT is indirect and cell type dependent. Moreover, functional analysis identifies cell adhesion-related genes that have been changed by hTERT in both cell lines. Adhesion experiments revealed that hTERT expression significantly increases cell adhesion. Monolayer wound healing and transwell assays demonstrated increased cell migration upon hTERT expression. These results provide new evidence to support a noncanonical function for hTERT in promoting tumorigenesis.
| S-EPMC4789728 | biostudies-literature
Project description:Telomere lengths are tightly regulated within a narrow range in normal human cells. Previous studies have extensively focused on how short telomeres are extended and have demonstrated that telomerase plays a central role in elongating short telomeres. However, much about the molecular mechanisms of regulating excessively long telomeres is unknown. In this report, we demonstrated that the telomerase enzymatic component, hTERT, plays a dual role in the regulation of telomere length. It shortens excessively long telomeres and elongates short telomeres simultaneously in one cell, maintaining the optimal telomere length at each chromosomal end for efficient protection. This novel hTERT-mediated telomere-shortening mechanism not only exists in cancer cells, but also in primary human cells. The hTERT-mediated telomere shortening requires hTERT's enzymatic activity, but the telomerase RNA component, hTR, is not involved in that process. We found that expression of hTERT increases telomeric circular DNA formation, suggesting that telomere homologous recombination is involved in the telomere-shortening process. We further demonstrated that shelterin protein TPP1 interacts with hTERT and recruits hTERT onto the telomeres, suggesting that TPP1 might be involved in regulation of telomere shortening. This study reveals a novel function of hTERT in telomere length regulation and adds a new element to the current molecular model of telomere length maintenance.
| S-EPMC4111723 | biostudies-literature
Project description:BackgroundTelomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated.Methodology/principal findingsTo understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function.Conclusions/significanceWe provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention.
| S-EPMC2744565 | biostudies-literature
Project description:Human telomerase reverse transcriptase is a ribonucleoprotein that synthesises telomeric sequences, which decrease at each cell division. In cancer cells, its activity is linked to telomere maintenance leading to unlimited cellular proliferation and immortality. To evaluate the prognostic value of the catalytic subunit telomerase reverse transcriptase (hTERT), we analysed its expression by immunohistochemistry in 122 formalin-fixed lung tumours including 42 squamous cell carcinoma (SCC), 43 adenocarcinoma (ADC), 19 basaloid carcinoma (BC) and 18 small-cell lung carcinoma (SCLC) in comparison with detection of hTERT mRNA by in situ hybridisation and relative telomerase activity by TRAP assay in a subset of tumours. We observed a high concordance between hTERT protein expression and detection of hTERT mRNA and telomerase activity. Telomerase expression varied according to histology (P=0.0002) being significantly lower in ADC than in SCC, BC and SCLC (P<0.0001). Adenocarcinoma and SCC exhibited either a nuclear or a nucleolar staining in contrast with a diffuse nuclear staining observed in most BC and all SCLC (P=0.01). In stage I NSCLC telomerase expression was lower than in other stages (P=0.04), and a nucleolar staining was correlated with a short survival (P=0.03). We concluded that telomerase expression and pattern are distinctive among histopathological classes of lung cancer and convey prognostic influence.
| S-EPMC2410220 | biostudies-other
Project description:BACKGROUND:Alterations in the insulin-like growth factor (IGF) system have been reported for different tumours. They are of particular interest in the search for new prognostic and therapeutic approaches in cancer. In childhood acute lymphoblastic leukaemia (ALL) the amount of "tumour mass" at diagnosis can exceed 1 kg. To understand the endocrine, paracrine, and autocrine potential of the malignant transformed progenitor cells, the ability of these cells to express components of the IGF system needs to be investigated. AIM:To characterise the expression pattern of genes of the IGF system in malignant lymphoblasts of children suffering from ALL. METHODS:Reverse transcription polymerase chain reaction of Ficoll separated mononuclear cells from 142 children with ALL, 127 cord blood samples, and 55 blood samples of age matched controls were studied. RESULTS:The expression of IGF-I, IGF-II, IGF binding protein 5 (IGFBP-5), and CTGF (IGFBP-rP2) was seen in a higher proportion of mononuclear cells of patients with ALL than in controls. Patients with ALL who were in continuous remission had a lower percentage of IGFBP-2 and IGFBP-3 expressing mononuclear cells at diagnosis than did those who developed a relapse. Only malignant lymphoblasts of B cell origin showed expression of CTGF (IGFBP-rP2). Malignant lymphoblasts of T cell origin more often expressed IGFBP-2 and IGFBP-5, whereas IGF-II and IGFBP-3 expression was seen more often in lymphoblasts of B cell origin. CONCLUSIONS:Malignant lymphoblasts of patients with ALL express components of the IGF system and therefore promote their own growth in an autocrine, paracrine, or endocrine manner. Whether these components will be useful as prognostic factors in the stratification of ALL treatment in children needs to be evaluated.
| S-EPMC1187145 | biostudies-literature
Project description:Acute myelogenous leukaemia (AML) is associated with risk factors that are largely unknown and with a heterogeneous response to treatment. Here, we provide a comprehensive quantitative understanding of AML proteomic heterogeneities and hallmarks by using the AML Proteome Atlas, a proteomics database that we have newly derived from MetaGalaxy analyses, for the proteomic profiling of 205 patients with AML and 111 leukaemia cell lines. The analysis of the dataset revealed 154 functional patterns based on common molecular pathways, 11 constellations of correlated functional patterns and 13 signatures that stratify the outcomes of patients. We find limited overlap between proteomics data and both cytogenetics and genetic mutations. Moreover, leukaemia cell lines show limited proteomic similarities with cells from patients with AML, suggesting that a deeper focus on patient-derived samples is needed to gain disease-relevant insights. The AML Proteome Atlas provides a knowledge base for proteomic patterns in AML, a guide to leukaemia cell line selection, and a broadly applicable computational approach for quantifying the heterogeneities of protein expression and proteomic hallmarks in AML.
| S-EPMC7051028 | biostudies-literature
Project description:We have mapped the 5' and 3' boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3' end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.
| S-EPMC84565 | biostudies-literature