Bioluminescent Method for Detecting Telomerase Activity

Xu, Shunqing; He, Min; Yu, Hongping; Wang, Xiaoyang; Tan, Xi; Lü, Bin; Sun, Xi; Zhou, Yikai; Yao, Qunfeng; Xu, Yongjun; Zhang, Zhiren

doi:10.1093/clinchem/48.7.1016

Cited by 20 publications

(15 citation statements)

References 23 publications

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“…2000; Yoshino et al . 2003), we quantified telomerase activity in neural precursors with an ELIPA, which correlates well with the common semi‐quantitative TRAP assay (Xu et al . 2002).…”

Section: Effects Of Low Oxygen On Telomerase Activitymentioning

confidence: 89%

“…When the telomere shortens to the critical stage, the intensity of cell division decreases significantly, and cells differentiate and age (Wynford-Thomas 1996). Although telomerase activity is usually evaluated by PCR-based telomeric repeat amplification protocol (TRAP assay) (Harada et al 2000;Yoshino et al 2003), we quantified telomerase activity in neural precursors with an ELIPA, which correlates well with the common semi-quantitative TRAP assay (Xu et al 2002). The PP i is quantitatively converted to ATP by ATP-sulfurylase and ATP is determined by the luciferase luminescence system.…”

Section: Effects Of Low Oxygen On Telomerase Activitymentioning

confidence: 95%

“…Cells were lysed with cytoplasmic extraction buffer [10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1.5 mM MgCl 2, 0.1% NP-40, Complete protease inhibitors (Roche)]. Telomerase assay was performed as described previously with minor modifications (Xu et al 2002). For the assay, 3 lL cell extract with a protein content of 5 lg/lL were used.…”

Section: Determination Of Telomerase Activity By Enzymatic Luminometrmentioning

confidence: 99%

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Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors

Milošević

Schwarz²,

Krohn

et al. 2005

Journal of Neurochemistry

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The efficient generation of specific brain cells in vitro may serve as a source of cells for brain repair in several devastating neurological diseases. Production of dopaminergic neurons from precursor cells for transplantation in Parkinson's disease has become a major research goal. We found that murine mesencephalic neurospheres were viable and proliferated, preserved telomerase activity, pluripotency and dopaminergic commitment for many weeks when cultured in 3% O 2 , whereas exposing these cells to 21% oxygen prohibited long-term expansion. Microarray data suggest that a variety of genes related to the cell cycle, cell maturation and apoptosis are differentially regulated in midbrain-derived precursors cultured in 3 versus 21% oxygen after 1-2 months. Taken together, we hypothesize that sustained high oxygen has deleterious effects on the self-renewal capacity of mesencephalic neural precursors, possibly accelerating maturation and senescence resulting in overall cell loss. Gene regulation governed by low oxygen tension may be relevant to the normal development and survival of midbrain neurons. Keywords: microarray, neural precursors, neurospheres, oxygen, proliferation, telomerase. Stem cells are undifferentiated cells that retain the ability to divide and proliferate throughout postnatal life to provide precursor cells that can differentiate into specialized cells. Neural stem cells can give rise to all the cells of the CNS including glial and neuronal cells (Gage 2003). As a result of their ability to differentiate following transplantation, these cells represent a potentially valuable therapeutic tool for the treatment of genetic, traumatic, inflammatory or degenerative neurological disorders (Studer et al. 1998;Storch and Schwarz 2002;Conti et al. 2003). To enable long-term expansion, all precursor cells must maintain the capacity to self-renew and a positive balance of proliferation versus differentiation or cell death. Therefore, it is crucial to understand the mechanisms governing cell division, differentiation and senescence (Sharpless and DePinho 2004).Mammals have evolved complex circulatory systems to ensure that every cell receives sufficient oxygen for normal metabolic processes. Lack of oxygen is defined as a reduction in oxygen supply to the tissues to below physiological levels. Hypoxia can develop as a result of ischaemia, representing a pathological component of stroke and heart attack. In addition to being a consequence of the growth of a malignant tumour and so a potential marker, hypoxia also acts to promote tumour development (Brizel et al. 1996;Brat et al. 2004). Address correspondence and reprint requests to Javorina Milosevic, Department of Neurology, Max-Bürger-Forschungszentrum, Johannisallee 30, 04103 Leipzig, Germany. E-mail: javorina.Milosevic@medizin.uni-leipzig.deAbbreviations used: ADA, adenosine deaminase; bFGF, basic fibroblast growth factor; BrdU, 5-bromo-2-deoxyuridine; DA, dopamine; DAPI, 6¢-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EGF...

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“…2000; Yoshino et al . 2003), we quantified telomerase activity in neural precursors with an ELIPA, which correlates well with the common semi‐quantitative TRAP assay (Xu et al . 2002).…”

Section: Effects Of Low Oxygen On Telomerase Activitymentioning

confidence: 89%

Section: Effects Of Low Oxygen On Telomerase Activitymentioning

confidence: 95%

Section: Determination Of Telomerase Activity By Enzymatic Luminometrmentioning

confidence: 99%

See 1 more Smart Citation

Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors

Milošević

Schwarz²,

Krohn

et al. 2005

Journal of Neurochemistry

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“…A luciferase system for bioluminescence detection and a luminometer are required [123] Electrochemiluminescence method Telomerase activity can also be determined by electrochemiluminescence (luminescence upon electrolysis). In this method, the 5 0 -biotinconjugated primer is elongated by telomerase followed by incubation with a suspension of magnetic beads modified with avidin This method allows for the quantitative determination of telomerase activity in samples that contain at least 500 HeLa cells.…”

Section: Description Of Modification or Original Assay Benefits Of Momentioning

confidence: 99%

Telomerase Structure and Function, Activity and Its Regulation with Emerging Methods of Measurement in Eukaryotes

Yadav¹,

Wakil²

2020

Telomerase and Non-Telomerase Mechanisms of Telomere Maintenance

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The telomerase reverse transcriptase has an essential role in telomere maintenance which is very important in aging process and cancer biology. Recent studies have revealed three-dimensional architecture of both human and ciliate telomerase at about 25 Å resolution, using single particle electron microscopy (EM). Telomerase supplements the tandem array of simple-sequence repeats at chromosome ends to compensate for the DNA erosion inherent in genome replication which makes it to be distinct among polymerases. Telomeres are found at the end of eukaryotic linear chromosomes and proteins that bind to them and help to protect DNA from being recognized as double-strand breaks thus preventing end-to-end fusions. The activity of telomerase is tightly regulated at multiple levels of cellular development, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere site for activation and processing. Commonly used methods in telomere biology are telomere restriction fragment (TRF), telomere repeat amplification protocol (TRAP) and telomere dysfunction induced foci (TIF) analysis. This chapter summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between different cell types and non-telomerase telomere maintenance mechanisms.

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“…Consequently, telomerase is a promising biomarker and a potential therapeutic target. The detection of telomerase activity is not only valuable for clinical tumour diagnosis and therapy but is also significant for developing telomerase-targeted drugs 11 12 13 .…”

mentioning

confidence: 99%

Coupling a DNA-Based Machine with Glucometer Readouts for Amplified Detection of Telomerase Activity in Cancer Cells

Wang

Huang

et al. 2016

Sci Rep

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The strong correlation between cancer and telomerase activity has inspired the development of new strategies to evaluate telomerase activity. Here, a personal glucose meter (PGM) system that uses DNA-based machine amplification to detect telomerase in cancer cells is reported. In this assay, telomerase elongation products are amplified in the form of another type of product by a DNA-based machine. This process can only be activated by the hybridization of the extended telomerase substrate (TS) probe and the complementary primer in the presence of telomerase. The obtained products are then transformed to glucose-related signals via a three-component assay, which enables the simple use of a PGM to indirectly quantify the telomerase activity. The proposed method realizes sensitive telomerase activity detection down to 20 HeLa cells with a significantly enhanced dynamic range. Additionally, short telomerase elongation products, such as telomerase substrate probes with two repetitive sequences, that cannot be detected using the most widely used telomeric repeat amplification protocol assay were detected.

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Bioluminescent Method for Detecting Telomerase Activity

Cited by 20 publications

References 23 publications

Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors

Low atmospheric oxygen avoids maturation, senescence and cell death of murine mesencephalic neural precursors

Telomerase Structure and Function, Activity and Its Regulation with Emerging Methods of Measurement in Eukaryotes

Coupling a DNA-Based Machine with Glucometer Readouts for Amplified Detection of Telomerase Activity in Cancer Cells

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