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Telomeric Repeat Amplification Protocol: Measuring the Activity of the Telomerase
Protocol First Online: 07 March This is a preview of subscription content, log in to check access. Kim NW, Wu F. Nucleic Acids Res. Burger AM.
Methods Mol Biol. PubMed Google Scholar. Telomeres serve to maintain chromosome integrity, preventing illegitimate recombination and end-to-end joining de Lange, ; Harrington, ; Blasco, If this loss is not compensated, the telomeres will reach a critically short length, triggering the cell to enter a state of replicative senescence or apoptosis McEachern et al. Telomerase is the ribonucleoprotein RNP enzyme that synthesizes telomeres. These two components are essential for the activity of the enzyme.
Human telomere synthesis occurs early in development Collins and Mitchell, ; Cong et al. The majority of adult somatic cells do not have appreciable telomerase activity and telomeres gradually shorten, limiting cell division capacity Harley et al. In the majority of human cancers, however, telomerase is reactivated and provides the sustained proliferative capacity of these cells Shay and Bacchetti, An understanding of telomerase biology thus has important implications for both cancer and aging.
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Telomeres are synthesized during S phase in human cells Ten Hagen et al. Some existing evidence is consistent with the idea that the cell cycle-dependent regulation of telomerase could occur at the level of subcellular trafficking. Redistribution of components of telomerase has been observed during S phase, when telomere synthesis occurs. Wong et al. In similar studies, Yang et al. Regarding the other essential component of the enzyme, Jady et al. However, although there is some evidence that both hTR and hTERT move during S phase, movement to telomeres, and thus a direct link to regulation of function, has not been found in vertebrate cells.
More persuasive evidence is available from ciliates, where telomerase RNA localizes to discrete nuclear foci throughout most of the cell cycle, and a fraction of the RNA is mobilized to the replication band, the site of DNA and telomere synthesis, during S phase Fang and Cech, In this work, we have investigated the subnuclear distribution of endogenous human TR and TERT over the course of the cell cycle and found compelling evidence that regulation of telomerase activity occurs via trafficking of hTR and hTERT in human cells.
Here, we provide the first clear evidence that hTERT resides in subnuclear foci that do not correspond to nucleoli, Cajal bodies, or telomeres during most of the cell cycle. In addition, we find that both hTR and hTERT localize to foci adjacent to Cajal bodies during S phase, marking a potential site outside of telomeres where both endogenous telomerase components are detected.
The implications of our findings with regard to telomerase biogenesis and telomere length regulation are discussed. Synchronous populations of HeLa cells were obtained by double thymidine block. Briefly, cells were treated with 2 mM thymidine Sigma-Aldrich for 18 h, released in normal media for 9 h, and retreated with 2 mM thymidine for another 18 h. Alternatively, HeLa cells were synchronized by treatment with 5 mM hydroxyurea for 24 h after 24 h of serum withdrawal.
Cells were fixed at various time points after release and analyzed as described below. FISH was performed essentially as described previously Zhu et al. Coverslips were mounted as described above. Cells were incubated with one or both of the following antibodies at the indicated dilution for 1 h at room temperature: mouse anti-p80 coilin ,; gift from G.
After three PBS washes, slides were mounted as described above.
All antibodies were diluted in PBST. Cells were washed in PBS and then fixed with chilled acetone for 5 min. After three PBS washes, the cells were mounted as described above. Both secondary antibodies were obtained from Jackson ImmunoResearch Laboratories. Our laboratory developed a FISH procedure specific for the detection of endogenous hTR in human cells and was the first to describe its intranuclear localization Zhu et al.whitelabel.tradetoolsfx.com/includes/hapej-meilleur-prix-azithromycine.php
Telomeres and Telomerase: Methods and Protocols (Methods in Molecular Biology Vol 191)
Using this technique, we found that hTR localized to Cajal bodies in telomerase-positive human cancer cell lines Zhu et al. In the course of that study performed with asynchronous populations of cells , we noticed that a small percentage of cells displayed different patterns of hTR localization. For example, we observed additional hTR foci that did not stain with antibodies against coilin the marker protein of Cajal bodies.
To investigate whether the secondary hTR localization patterns observed at low frequency in asynchronous populations reflected cells in particular stages of the cell cycle, we performed hTR FISH and coilin IF on cell cycle-synchronized HeLa cervical carcinoma cells. HeLa cells were synchronized with a double thymidine block or hydroxyurea; our unpublished data and monitored for synchronization efficiency at the indicated time points by BrdU analysis to distinguish S-phase cells and DAPI staining to distinguish mitotic cells.
Fluorescence-activated cell sorting analysis with propidium iodide-stained cells to examine DNA content was also performed to confirm the synchronization efficiency in some cases. The BrdU staining patterns also allowed us to assess the S subphase i. As expected, throughout the majority of the cell cycle, hTR localizes exclusively to Cajal bodies Figure 1 , G 1 and G 2.
However, during S phase, a dynamic change in the subcellular distribution of hTR was observed in a significant number of cells. In most cells, coilin colocalized with hTR in the rings, as shown Figure 1 , row 2. By mid-S phase, the distribution of hTR foci in rings had declined, and hTR occurred in small, nucleoplasmic foci that did not stain with anti-coilin antibodies Figure 1 , row 3, denoted by arrowheads. In addition, hTR was sometimes observed in foci immediately adjacent to Cajal bodies detailed below. These patterns were specific to S phase; they were not observed in cells in G 1 or G 2 phases of the cell cycle.
Figure 1. Differential interference contrast DIC panels show differential interference light microscopy data.
Telomeres and Telomerase: Methods and Protocols - Google книги
Merge panels show superimposition of hTR and coilin fluorescence data yellow indicates overlap of signal. Arrows in mid-S merge panel indicate hTR foci that do not colocalize with coilin. During mitosis, previous research has shown that Cajal bodies dissociate, and then reform during early G 1 of the cell cycle Andrade et al.
We found that hTR localization parallels that of the Cajal body marker protein coilin during cell division, with only a slight temporal delay in reassociation after mitosis. Like coilin, hTR displayed a diffuse localization pattern throughout mitosis Figure 1 , row 5 shows hTR diffusely localized in interchromatin region.
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No association with discrete foci was observed. After mitosis, Cajal body formation precedes hTR accumulation in Cajal bodies. Together, these results demonstrate that the cell cycle has dramatic effects on the localization of hTR and that there is a dynamic, S phase-specific rearrangement of hTR in human cancer cell nuclei. We found that the ring-like pattern of hTR and coilin foci observed during early S phase seems to correspond to localization to the periphery of nucleoli Figure 2A.
In addition to the ring pattern, we also observed hTR foci that seemed to be distributed across the surface of a nucleolus in some focal planes Figure 2A. In the majority of cases, the hTR ring was observed around only one nucleolus within a given cell, suggesting a previously undescribed heterogeneity among individual nucleoli. The localization of Cajal bodies to the periphery of nucleoli is well documented indeed, Cajal bodies were initially termed nucleolar accessory bodies; Gall, ; Cioce and Lamond, , although an S-phase-specific association has not been described previously.
We cannot exclude the possibility that the Cajal bodies and hTR are internal to nucleoli based on the studies presented here, but the distribution of the foci is suggestive of localization on the surface of the nucleolus. Elegant studies in HeLa cells have demonstrated the movement of Cajal bodies to and from nucleoli Platani et al. Our results suggest that Cajal bodies containing hTR move to the periphery of nucleoli during early S phase in HeLa cells.
To ascertain whether the coilin-negative, S-phase-specific hTR foci described above Figure 1 , row 3 were at telomeres, we coanalyzed hTR and telomeres in S-phase cells.
During mid-S, there was an increase in both the number of cells that had hTR-telomere associations and in the number of associations per cell. These results indicate that human telomerase RNA moves to telomeres during mid-S phase. Here, we have investigated the localization of endogenous hTERT protein. The staining pattern described previously for this antibody in HeLa cells was more generally nucleoplasmic Masutomi et al.
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We found that the more restricted staining pattern that we obtained was specific to hTERT. We observed some staining in the primary fibroblasts Figure 3A , consistent with previous observations that normal cells express a low level of hTERT protein Masutomi et al. The number and intensity of the foci was significantly greater in the cells expressing exogenous hTERT. Figure 2. Human telomerase RNA is found at the periphery of nucleoli and at telomeres during S phase.
A hTR and coilin seem to be associated with the surface of nucleoli during early S phase. The cells shown are in early S phase. Arrowheads denote nucleoli exhibiting apparent peripheral or surface hTR signals. B hTR associates with telomeres during mid-S phase. BrdU panel shows a mid-S-phase pattern. Arrows in merge panels indicate foci where both hTR and telomeres are present.
The results suggest an S-phase-specific mobilization of the components of the telomerase RNP to its functional destination, the telomere. Figure 3. Cellular localization of human telomerase reverse transcriptase. Data in each pair of panels were normalized to allow visual comparison. Figure 4. Temporal patterns of association of human telomerase RNA and telomerase reverse transcriptase with nuclear structures during S phase in HeLa cells.