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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Elevated telomerase activity is an important molecular signature of cancer cells and primitive cells in regenerative tissues. However, isolation of single living cells with endogenous telomerase activity has not yet been possible. Here, we developed adenovirus serotype 35 tropism-based vectors encoding destabilized enhanced green fluorescence protein with a half-life of 2 h (d2EGFP) driven by the human telomerase reverse transcriptase (hTERT) promoter. As assessed in telomerase-positive or -negative cell lines, the d2EGFP expression positively correlated with hTERT transcript content and telomerase activity. In retinoic acid-induced differentiating HL-60 cells, the d2EGFP expression is diminished in the same manner as the hTERT expression. Individual cells from HeLa and HL-60 cell lines exhibited heterogeneous d2EGFP expression, which was cell cycle dependent, as the sorted d2EGFP+ HL-60 cells contained twice as many cells in S/G2/M phase of the cell cycle compared with the d2EGFP- HL-60 cells. However, both cell populations exhibited the same proliferation and regeneration capacities. Heterogeneous d2EGFP expression was also detected in xenograft glioblastoma multiforme cells with tumor formation capacity. Thus, d2EGFP expression reported cell cycle- and differentiation stage-dependent hTERT expression. Our study facilitates isolation and characterization of single living cells with telomerase activity.
Mol Ther 2006 Jul
PMID:Detection of cell cycle- and differentiation stage-dependent human telomerase reverse transcriptase expression in single living cancer cells. 1658 24

Chromatin, the eukaryotic template of genetic information, is subject to a diverse array of posttranslational modifications that largely impinge on the N-termini of histones, such as acetylation, methylation, phosphorylation, and ubiquitination. Distinct histone modifications generate synergistic or antagonistic interaction affinities for novhistone proteins, which in turn dictate dynamic transitions between transcriptionally active or silent states of chromatin. Besides transcription, numerous biological processes, including DNA replication, DNA repair, and recombination, are regulated by chromatin-associated factors. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between the structural or regulatory proteins and DNA and its role in regulating diverse cellular processes in vivo by formaldehyde crosslinking of proteins to proteins and proteins to DNA, followed by immunoprecipitation of the fixed material and detection of the associated DNA. Here we illustrate the overall experimental procedure by taking ChIP analysis of the human telomerase reverse transcriptase promoter as an example.
Methods Mol Biol 2006
PMID:Histone modifications and transcription factor binding on chromatin ChIP-PCR assays. 1676 33

Human telomerase reverse transcriptase (hTERT) is a telomerase catalytic subunit that regulates telomerase activity. Telomerase is expressed in many human cancers and cell lines and is thought to contribute to their immortality. Little is known about the expression of telomerase in non-epithelial tumors. The objective of this study was to evaluate hTERT expression in a wide range of soft tissue sarcomas. A total of 154 cases of different types of soft tissue sarcoma (54 low-grade, 40 intermediate-grade, and 60 high-grade cases) were evaluated for hTERT expression using immunohistochemistry on tissue microarrays. hTERT immunoexpression was detected in 59% of cases; it was observed in 46%, 58%, and 72% of low-grade, intermediate-grade, and high-grade sarcoma cases, respectively. The intensity of staining positively correlated with the grade of the sarcomas: diffuse strong positive nuclear staining was identified in 6, 8, and 30 cases of low-grade, intermediate-grade, and high-grade sarcomas, respectively. These results suggest that telomerase expression is more often detected in highly malignant tumors than in low-grade sarcomas and thus may be a critical mechanism in tumor progression.
Appl Immunohistochem Mol Morphol 2006 Jun
PMID:Immunohistochemical detection of hTERT protein in soft tissue sarcomas: correlation with tumor grade. 1678 90

Breast cancer is the most common malignancy among women. Current therapies for breast tumors are based on the use of chemotherapeutic drugs that are quite toxic for the patients and often result in resistance. Telomerase is up-regulated in 95% of breast carcinomas but not in adjacent normal tissues. Therefore, it represents a very promising target for anticancer therapies. Unfortunately, the antiproliferative effects of telomerase inhibition require extensive telomere shortening before they are fully present. Combining telomerase inhibition with common chemotherapeutic drugs can be used to reduce this lag phase and induce tumor cell death more effectively. Few studies have analyzed the effects of telomerase inhibition in combination with anticancer drugs in breast cancer cells. In this study, we inhibited telomerase activity in two breast cancer cell lines using a dominant-negative human telomerase reverse transcriptase and analyzed cell viability after treatment with different anticancer compounds. We found that dominant-negative human telomerase reverse transcriptase efficiently inhibits telomerase activity and causes telomere shortening over time. Moreover, cells in which telomerase was suppressed were more sensitive to anticancer agents independently of their mechanism of action and this sensitization was dependent on the presence of shorter telomeres. Altogether, our data show that blocking telomere length maintenance in combination with anticancer drugs can be used as an effective way to induce death of breast cancer cells.
Mol Cancer Ther 2006 Jul
PMID:Telomerase inhibition enhances the response to anticancer drug treatment in human breast cancer cells. 1689 52

A major goal in cancer gene therapy is to develop efficient gene transfer protocols that allow tissue-specific and tightly regulated expression of therapeutic genes. The ideal vector should efficiently transduce cancer cells with minimal toxicity on normal tissues and persistently express foreign genes. One of the most promising regulatory systems is the mifepristone/RU486-regulated system, which has much lower basal transcriptional activity and high inducibility. In this work, we modified this system by incorporating a cancer-specific promoter, the human telomerase reverse transcriptase (hTERT) promoter. By utilizing hTERT promoter to control the regulator, RU486 could specifically induce the expression of foreign genes in cancer cells but not in normal cells. In the context of this system, a dominant negative mutant of survivin (surDN) was controllably expressed in colorectal tumor cells. The surDN expression induced by RU486 showed a dosage- and time-dependent pattern. Regulated expression of surDN caused caspase-dependent apoptosis in colorectal tumor cells but had little effect on normal cells. Analysis of cell viability showed that RU486-induced expression of surDN suppressed colorectal tumor cell growth and had synergic effect in combination with chemotherapeutic agents. The potential of this system in cancer therapy was evaluated in experimental animals. Tumor xenograft models were established in nude mice with colorectal tumor cells, and RU486 was intraperitoneally administered. The results showed that conditional expression of surDN efficiently inhibited tumor growth in vivo and prolonged the life of tumor-burdened mice. Synergized with the chemotherapeutic drug cisplatin, regulated surDN expression completely suppressed tumor growth. These results indicated that this modified RU486-regulated system could be useful in cancer-targeting therapy.
J Mol Med (Berl) 2006 Dec
PMID:Suppression of colorectal tumor growth by regulated survivin targeting. 1707 82

The human telomerase reverse transcriptase hTERT is highly expressed in undifferentiated embryonic cells and silenced in the majority of somatic cells. To investigate the mechanisms of hTERT silencing, we have developed a novel reporter using a bacterial artificial chromosome (BAC) that contained the entire hTERT gene and its neighboring loci, hCRR9 and hXtrp2. Firefly and Renilla luciferases were used to monitor transcription from the hTERT and hCRR9 promoters, respectively. In mouse embryonic stem cells stably integrated with the BAC reporter, both hTERT and hCRR9 promoters were highly expressed. Upon differentiation into embryoid bodies and further into mineral-producing osteogenic cells, the hTERT promoter activity decreased progressively, whereas the hCRR9 promoter remained highly active, both resembling their endogenous counterparts. In fully differentiated cells, the hTERT promoter was completely silenced and adopted a chromatin structure that was similar to its native counterpart in human cells. Inhibition of histone deacetylases led to the opening of the hTERT promoter and partially relieved repression, suggesting that histone deacetylation was necessary but not sufficient for hTERT silencing. Thus, our result demonstrated that developmental silencing of the human TERT locus could be recapitulated in a chromosomal position-independent manner during the differentiation of mouse embryonic stem cells.
Mol Biol Cell 2007 Feb
PMID:Transcriptional silencing of a novel hTERT reporter locus during in vitro differentiation of mouse embryonic stem cells. 1715 55

Telomerase replenishes the telomeric repeats that cap eukaryotic chromosome ends. To perform DNA synthesis, the active site of telomerase reverse transcriptase (TERT) copies a template within the integral telomerase RNA (TER). In vivo, TERT and TER and additional subunits form a telomerase holoenzyme capable of telomere elongation. We previously purified epitope-tagged Tetrahymena thermophila TERT and characterized two of the associated proteins. Here we characterize the remaining two proteins that were enriched by TERT purification. The primary sequence of the p75 polypeptide lacks evident homology with other proteins, whereas the p20 polypeptide is the Tetrahymena ortholog of a conserved multifunctional protein, Skp1. Genetic depletion of p75 induced telomere shortening without affecting the accumulation of TER or TERT, suggesting that p75 promotes telomerase function at the telomere. Affinity purification of p75 coenriched telomerase activity and each other known telomerase holoenzyme protein. On the other hand, genetic depletion of Skp1p induced telomere elongation, suggesting that this protein plays a negative regulatory role in the maintenance of telomere length homeostasis. Affinity purification of Skp1p did not detectably enrich active telomerase but did copurify ubiquitin ligase machinery. These studies reveal additional complexity in the positive and negative regulation of Tetrahymena telomerase function.
Mol Cell Biol 2007 Mar
PMID:Positive and negative regulation of Tetrahymena telomerase holoenzyme. 1722 Feb 81

Telomerase activation is a critical event in cell immortalization, and an increase in human telomerase reverse transcriptase (hTERT) expression is the key step in activating telomerase. The phosphatase and tensin homolog (PTEN) gene encodes a double-specific phosphatase that induces cell cycle arrest, inhibits cell growth, and causes apoptotic cell death. Here, we evaluated a combined PTEN and antisense hTERT gene therapy for experimental glioma in vitro and in vivo. We demonstrated that infection with antisense-hTERT and wild-type-PTEN adenoviruses significantly inhibited human U251 glioma cell proliferation in vitro and glioma growth in a xenograft mouse model. The efficacy of therapy was obviously higher in the tumor xenografts infected with both PTEN and antisense hTERT than in the gliomas infected with either agent alone at the same total viral dose. Consistent with these results, we showed that telomerase activity and hTERT protein levels were markedly reduced in the glioma cells following adenovirus infection. In contrast, the levels of PTEN protein expression were dramatically increased in these cells. Our data indicate that combination treatment with antisense hTERT and wild-type PTEN effectively suppresses the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach in glioma gene therapy that warrants further investigation.
Cell Mol Life Sci 2007 Mar
PMID:Evaluation of combination gene therapy with PTEN and antisense hTERT for malignant glioma in vitro and xenografts. 1731 Feb 80

Physiological hypoxia extends the replicative life span of human cells in culture. Here, we report that hypoxic extension of replicative life span is associated with an increase in mitochondrial reactive oxygen species (ROS) in primary human lung fibroblasts. The generation of mitochondrial ROS is necessary for hypoxic activation of the transcription factor hypoxia-inducible factor (HIF). The hypoxic extension of replicative life span is ablated by a dominant negative HIF. HIF is sufficient to induce telomerase reverse transcriptase mRNA and telomerase activity and to extend replicative life span. Furthermore, the down-regulation of the von Hippel-Lindau tumor suppressor protein by RNA interference increases HIF activity and extends replicative life span under normoxia. These findings provide genetic evidence that hypoxia utilizes mitochondrial ROS as signaling molecules to activate HIF-dependent extension of replicative life span.
Mol Cell Biol 2007 Aug
PMID:Mitochondrial reactive oxygen species trigger hypoxia-inducible factor-dependent extension of the replicative life span during hypoxia. 1756 66

Tumor-associated human telomerase reverse transcriptase (hTERT) is expressed in >85% of human tumors but not in most normal cells. As a result, this antigen has received considerable attention from those interested in cancer immunotherapy. Specifically, there has been strong interest in MHC class I-associated peptides derived from hTERT because these are expressed on the cell surface and thus may enable the targeting of tumor cells. Much of this interest has focused on peptide 540-548, ILAKFLHWL, which was predicted to exhibit the strongest binding to the common HLA A*0201 presenting molecule. The hTERT(540-548) peptide is currently being assessed in therapeutic vaccination trials; however, there is controversy surrounding whether it is naturally processed and presented on the surface of neoplastic cells. Here, we generate two highly sensitive reagents to assess the presentation of hTERT(540-548) on tumor cells: (a) a CD8(+) CTL clone, and (b) a recombinant T-cell receptor (TCR) that binds with picomolar affinity and a half-life exceeding 14 h. This TCR enables the identification of individual HLA A2-hTERT(540-548) complexes on the cell surface. The use of both this TCR and the highly antigen-sensitive CTL clone shows that the hTERT(540-548) peptide cannot be detected on the surface of tumor cells, indicating that this peptide is not a naturally presented epitope. We propose that, in future, rigorous methods must be applied for the validation of peptide epitopes used for clinical applications.
Mol Cancer Ther 2007 Jul
PMID:The HLA A*0201-restricted hTERT(540-548) peptide is not detected on tumor cells by a CTL clone or a high-affinity T-cell receptor. 1762 Apr 37


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