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First published online 11 March 2008
doi: 10.1242/jcs.019372
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Research Article |
1 Crucible Laboratory, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, NE4 6BE, UK
2 Henry Wellcome Biogerontology Laboratory and Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, NE4 6BE, UK
3 School of Clinical and Laboratory Sciences, Newcastle University, Newcastle upon Tyne, NE4 6BE, UK
4 Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, NE4 6BE, UK
* Author for correspondence (e-mail: t.vonzglinicki{at}ncl.ac.uk)
Accepted 17 January 2008
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Summary |
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 Top Summary IntroductionResultsDiscussionMaterials and MethodsReferences |
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Key words: Telomerase, TERT, Mitochondria, Oxidative stress, Reactive oxygen
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Introduction |
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). In addition, there is evidence for telomere length-independent functions of telomerase, which appear to promote cell survival and stress resistance (del Bufalo et al., 2005; Kang et al., 2004; Ludwig et al., 2001; Oh et al., 2001; Zhang et al., 2003). For example, telomerase expression conferred increased resistance to specific DNA damaging agents (Kondo et al., 1998; Ludwig et al., 2001; Sharma et al., 2003) and decreased apoptosis (Fu et al., 2000; Massard et al., 2006; Zhang et al., 2003; Zhu et al., 2000). Telomerase transgenic and knockout mice have been used to discriminate between telomere-dependent and -independent functions of telomerase. Increased metabolic turnover and augmented antioxidant responses in germ line cells (Franco et al., 2005) and increased sensitivity to chemotherapeutic (Lee et al., 2001) or alkylating (Gonzalez-Suarez et al., 2003) agents have been related to late generation Terc–/– mice specifically, suggesting a predominant dependency of these phenotypes on the telomere capping state in this model. By contrast, overexpression of mouse TERT confers increased stress resistance, improved antioxidant defence and improved differentiation capacity to mouse embryonic stem cells (Armstrong et al., 2005), and promotes proliferation of resting stem cells in the skin epithelium in a telomere-independent manner (Sarin et al., 2005). Tert–/– mice also show reduced glutamate+malate (complexI-dependent) respiration of heart mitochondria (J. Haendeler et al., unpublished).
The cell protective function of telomerase has been related to improved DNA damage repair (Sharma et al., 2003) or to increased apoptosis resistance (Fu et al., 2000; Massard et al., 2006; Zhang et al., 2003; Zhu et al., 2000). A better understanding of the mechanisms involved is of obvious importance, as telomerase is active in the vast majority of human cancers (Shay and Bacchetti, 1997) and might significantly contribute to tumorigenic potential (Bagheri et al., 2006; Stewart et al., 2002) and to the low sensitivity of many cancer cells to chemo- and radiotherapeutic treatments (Bakalova et al., 2003).
It had been shown recently that telomerase is excluded from the nucleus upon oxidative stress (Haendeler et al., 2003; Haendeler et al., 2004; Santos et al., 2004; Santos et al., 2006b). Santos et al. have found a mitochondrial localisation signal in the TERT sequence that directs the protein to mitochondria and have demonstrated a mitochondrial localisation for TERT (Santos et al., 2004). Stress-dependent nuclear exclusion has been demonstrated not only for ectopically overexpressed TERT, but also for wild-type TERT in endothelial cells (Haendeler et al., 2004), indicating that this shuttling is not an overexpression artefact. Possible biological function(s) of telomerase in an extra-nuclear localisation remained unclear, however. Liu et al. have described phosphorylation-dependent nuclear import as a method for regulation of telomerase activity in human lymphocytes (Liu et al., 2001). Santos et al. found an aggravation of mitochondrial DNA (mtDNA) damage in cells expressing TERT (Santos et al., 2004; Santos et al., 2006b).
By contrast, we describe here improved mitochondrial function, including better protection of mtDNA together with export of TERT towards mitochondria. We used chronic mild oxidative stress to explore the roles of an TERT transgene in MRC-5 lung fibroblasts for both telomere maintenance and stress resistance. Telomerase did not prevent telomere shortening under hyperoxia as it translocated gradually from the nucleus to mitochondria. However, TERT overexpression reduced mtDNA damage levels under oxidative stress and improved mitochondrial function, e.g. resulted in lower mitochondrial production of reactive oxygen species (ROS) together with an enhanced mitochondrial membrane potential (MMP). This was accompanied by indicators for a reduced retrograde response in terms of gene expression pattern and mitochondrial biogenesis. We suggest that protection of mitochondria against oxidative stress is an important telomere length-independent function for telomerase in cell survival.
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Results |
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). Increasing the ambient oxygen concentration to 40% (hyperoxia) generates increased peroxide levels and shortens the replicative lifespan of primary human fibroblasts because of stress-induced accelerated telomere shortening (Passos et al., 2007; von Zglinicki, 2002). Net growth of MRC5-TERT cells under hyperoxia decreased as in control MRC5 cells under the same conditions, albeit somewhat slower (data not shown). Growth arrest was confirmed by loss of expression of Ki67, a marker for cell proliferation, as well as increased frequencies of cells positive for senescence-associated β-galactosidase (SA-β-Gal) or phosphorylated histone H2A variant X (
-H2A.X), both markers for cellular senescence (d'Adda di Fagagna et al., 2003; Dimri et al., 1995; von Zglinicki et al., 2005). Although expression of TERT delayed the occurrence of these senescence markers under hyperoxia, eventually MRC5-TERT cells showed maximal marker expression similar to senescent parental cells (supplementary material Fig. S1).
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Under hyperoxia, telomere restriction fragment length in MRC5-TERT cells decreased (Fig. 1A) with the same rate as in parental MRC5 cells (Fig. 1B). This was confirmed by fluorescence in situ hybridization (FISH; Fig. 1C): metaphases from TERT-overexpressing fibroblasts under hyperoxia showed the same telomere loss as those from parental MRC5 cells (Fig. 1D). Moreover, frequencies of chromosomal end-to-end fusions were very similar in MRC5 and MRC5-TERT cells after prolonged hyperoxia (Fig. 1E). Thus, overexpression of TERT is sufficient to maintain telomere length in MRC5 fibroblasts growing under 21% oxygen, but not under 40% oxygen.
Growth arrest of TERT-overexpressing cells under oxidative stress is telomere-dependent but reversible
Colocalisation of telomeres and -H2A.X-containing DNA damage is a hallmark of telomere-dependent senescence (d'Adda di Fagagna et al., 2003). Telomere-induced DNA damage foci were found at similar frequencies in both MRC5 and MRC5-TERT cells following long-term culture under hyperoxia (Fig. 2A,B). Quantitatively, the correlation between telomeres and DNA damage foci was as good in MRC5-TERT cells as in senescent parental MRC5 (Fig. 2C).
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) and remains irreversible even if the cells are set back into normoxia, because short telomeres cannot be re-elongated. However, if MRC5-TERT cells were transferred back to normoxic conditions after a period of hyperoxia, they resumed growth, the frequency of Ki67-positive cells increased, cells lost H2A.X staining and telomeres became re-elongated (supplementary material Fig. S2), indicating that telomerase regains the ability to re-elongate telomeres even after long periods of increased oxidative stress.
TERT is excluded from nuclei under oxidative stress and accumulates in mitochondria
Total activity of telomerase in MRC5-TERT cells as measured by TRAP assay was not decreased under hyperoxia (Martin-Ruiz et al., 2004) (and data not shown). However, telomerase can be excluded from the nucleus in parallel with ROS generation (Haendeler et al., 2003; Haendeler et al., 2004). Telomerase activity as measured by Telomere Repeat Amplification Protocol (TRAP) in the extranuclear fraction (including mitochondria) increased from about 25-30% of the total cell activity in controls to 80–90% after both H2O2 treatment (Fig. 3A) and under hyperoxia (Fig. 3B). This was mostly due to an increase of TRAP activity in mitochondria and a corresponding decrease of nuclear TRAP activity (Fig. 3C).
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), we confirmed concentration-dependent H2O2-mediated nuclear exclusion of ectopically expressed TERT (Fig. 3D) and gradual nuclear exclusion of TERT under chronic hyperoxia (Fig. 3E). Western blotting of mitochondrial and nuclear fractions confirmed an increase of TERT protein specifically in mitochondria following treatment with hydrogen peroxide (supplementary material Fig. S3). Exclusion of TERT from the nuclei was reversible: when cells were shifted back from hyperoxia to normoxia, control conditions were restored after about 4 weeks (Fig. 3F). This time course corresponded well to the re-elongation of telomeres and resumption of cell growth.
TERT immunofluorescence in the cytoplasm significantly (Pearson's correlation coefficient P=0.98) colocalised with Mitotracker Green staining (Fig. 3G), thus confirming mitochondrial localisation of TERT in accordance with earlier reports (Santos et al., 2004; Santos et al., 2006b).
TERT protects mitochondria
One group recently reported higher susceptibility of mtDNA to damage in TERT-overexpressing fibroblasts (Santos et al., 2004; Santos et al., 2006b). We could not confirm this. On the contrary, we found that mtDNA in MRC5-TERT was protected against acute (Fig. 4A) or chronic (Fig. 4B) oxidative damage. We noted that mtDNA damage levels actually decreased under long-term hyperoxia, i.e. when most of the TERT protein (Fig. 3E) and telomerase activity (Fig. 3C) was translocated to mitochondria. This result was confirmed using an independent technique that measured damage via its effect on the supercoiled state of mtDNA (supplementary material Fig. S4).
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) (supplementary material Fig. S5). No significant differences were found between parental and empty vector (pBabe)-transfected cells (data not shown). Thus, TERT expression diminishes mitochondrial superoxide production and cellular ROS levels.
Mitochondrial membrane potential (MMP) is a direct indicator of the efficiency of coupling between respiration and ATP production. However, high MMP increases the probability of ROS generation (Balaban et al., 2005), and MMP in human fibroblasts responds to cellular ROS levels in an adaptative fashion, so that high ROS levels transcriptionally upregulate the uncoupling protein UCP2, resulting in MMP downregulation (Passos et al., 2007). Measuring MMP in intact cells by JC1 fluorescence we found higher membrane potential in MRC5-TERT cells (Fig. 4E). UCP2 expression was elevated by hyperoxia, but less so in MRC5-TERT cells (Fig. 4F). These data are in accordance with the idea that overexpression of TERT improves mitochondrial function, allowing low ROS levels despite tight coupling of mitochondria.
To test whether endogeneous TERT might also protect mitochondria, we knocked down endogeneous TERT in primary human umbilical vein endothelial cells (HUVECs) using two separate siRNAs (Fig. 4G). Mitochondrial superoxide production as measured by MitoSOX fluorescence and cellular peroxides measured by DHR fluorescence were significantly increased 2 days after transfection with both siRNAs in combination (Fig. 4H).
TERT overexpression counteracts retrograde response
Mitochondrial dysfunction induces major adaptative changes in global gene expression patterns often termed retrograde response, both in yeast (Jazwinski, 2005) and mammalian cells (Biswas et al., 2005), and this is also part of replicative senescence of human fibroblasts (Passos et al., 2007). In retrograde signalling, lower MMP results in the activation of cytoplasmic Ca2+ signalling, leading to metabolic readjustment, activation of mitochondrial biogenesis (Biswas et al., 2005; Passos et al., 2007) and downregulation of apoptotic signals. Having seen that TERT overexpression was able to improve mitochondrial function, we wanted to find out whether TERT would also downregulate retrograde response. TERT-overexpressing fibroblasts showed lower mitochondrial mass per cell under both normoxia and hyperoxia (Fig. 5A), mtDNA copy number was lower in MRC5-TERT cells under normoxia, and it did not increase under hyperoxia (Fig. 5B), altogether indicating diminished mitochondrial biogenesis.
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We recently identified 92 genes (120 Affymetrix probesets) with functions in cellular signalling, glycolysis and Krebs cycle metabolism, mitochondrial function and stress response, which were differentially expressed in senescent when compared with young MRC5 fibroblasts and, thus, might be candidate marker genes for retrograde response in human fibroblasts (Passos et al., 2007). Many of these genes changed expression in the opposite direction in MRC5-TERT when compared with senescent MRC5, while the majority of the remaining genes were not differentially expressed between young MRC5 and MRC5-TERT cells (Fig. 5C-F). Expression levels for nine selected genes were also evaluated by RT-PCR, and differential regulation was confirmed for all of them (supplementary material Fig. S6). In particular, we noted that many genes coding for enzymes with major functions in metabolism, including GLS, AK3, PDK4, RODH, GCLM and MAOA, were upregulated in senescence, but downregulated in MRC5-TERT cells (Fig. 5D,E). The same was true for genes involved in cellular signalling, including IGFBP3, SULF1 and BAMBI, while some signalling genes (e.g. ADCY3, AURKB, GPSM2) that were downregulated in senescence became upregulated following TERT overexpression (Fig. 5C). Interestingly, relatively few apoptosis-related genes were regulated in opposite fashion by senescence and TERT overexpression (ASC, BCOR and PHLDA1; Fig. 5F). Accordingly, we found that MRC5-TERT cells were significantly more resistant against apoptosis induced by either hydrogen peroxide (Fig. 5G) or etoposide (Fig. 5H).
Together, these data suggest that TERT overexpression lowers cellular ROS production, improves mitochondrial coupling, counteracts retrograde response and thus `rejuvenates' fibroblasts in terms of mitochondrial function. This is associated with improved resistance to apoptosis.
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Discussion |
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) and endogenous chronic oxidative stress in senescing endothelial cells (Haendeler et al., 2004) induced the export of TERT from the nucleus. So far, the effect of nuclear exclusion of TERT on telomere maintenance has not been examined intensively, although it had been noted that increased oxidative stress accelerated telomere shortening not only in human fibroblasts (von Zglinicki, 2002) but also in telomerase-positive endothelial cells (Furumoto et al., 1998; Kurz et al., 2004). Our data now show that chronic oxidative stress interferes with telomere maintenance at two levels: it increases the basal rate of telomere shortening by induction of telomeric DNA damage, as shown before in telomerase-negative fibroblasts (Petersen et al., 1998), and it prevents telomerase from counteracting telomere shortening by inducing its export from the nucleus and to mitochondria.
In terms of the effect of TERT on mtDNA integrity, our results are contrary to those published recently by one other group. Santos et al. (Santos et al., 2004; Santos et al., 2006b) have reported that ectopically expressed TERT in human fibroblasts under acute oxidative stress resulted in increased mtDNA damage. At present, we cannot resolve this discrepancy to our data. However, we note that our data not only demonstrate a protective effect of TERT on mtDNA integrity, but that we also find decreased mitochondrial ROS generation and increased MMP under both acute and chronic oxidative stress conditions, as well as indications for diminished retrograde response and improved apoptosis resistance, all demonstrating improved mitochondrial function in TERT-overexpressing cells. Our results are in accordance with recent data showing protection of mtDNA from UV- and ethidium bromide-mediated damage, as well as protection of mitochondrial function due to TERT expression (J. Haendeler et al., unpublished). Importantly, the fact that knock-down of TERT in HUVECs (this paper) as well as knock out of mouse TERT in mouse heart (J. Haendeler et al., unpublished) resulted in increased mitochondrial production of superoxide indicates that the mitochondrial effects of TERT are not simply overexpression artefacts, but describe a physiological function of telomerase. Kang et al. also found increased MMP and enhanced Ca2+ storage capacity in mouse neurons overexpressing mouse TERT (Kang et al., 2004). Moreover, in a model of apoptosis induction by BCL2 inhibition, TERT overexpression protected mitochondrial membrane potential and function independently of its catalytic activity with respect to telomere maintenance (del Bufalo et al., 2005).
The protective effect of telomerase against a variety of apoptosis- or senescence-inducing stressors has been related to TERT-mediated regulation of expression of genes implicated in cell proliferation and differentiation (Geserick et al., 2006), to improved DNA damage repair (Sharma et al., 2003; Smith et al., 2003), increased apoptosis resistance (del Bufalo et al., 2005; Zhang et al., 2003) or decreased apoptosis signalling (Dudognon et al., 2004). We show here evidence for the existence of a candidate protective mechanism that might be able to integrate some of these observations into a common concept: cells that overexpress TERT show evidence for improved mitochondrial function, specifically less mitochondrial superoxide production and lower levels of cellular ROS, improved mitochondrial coupling and suppressed retrograde response. An effect of telomerase on mitochondrial gene expression and function has been noted before in knock-down experiments in yeast (Nautiyal et al., 2002) and in mouse melanoma cells (Bagheri et al., 2006), although the direction and magnitude of the effect might be modified in tumour cells by their dependency on glycolysis as well as on telomerase (Li et al., 2005). Retrograde response has been described as a major reprogramming of nuclear gene expression patterns, including genes involved in metabolism, stress response and growth signalling, as result of mitochondrial dysfunction and resultant Ca2+-dependent signalling (Biswas et al., 2005; Butow and Avadhani, 2004). It is important to note in this respect that mitochondrial dysfunction and retrograde response is a characteristic feature of replicative senescence in both yeast (Jazwinski, 2005) and human fibroblasts (Passos et al., 2007). Thus, the effect of ectopic TERT expression can be described as a `rejuvenation' of mitochondria that impacts on global gene expression pattern via suppression of mitochondrial dysfunction and, possibly, retrograde signalling.
Our results suggest that mitochondrial location of TERT might be necessary for its protection of mitochondrial function. It has recently been shown that TERT binds to mtDNA (J. Haendeler et al., unpublished). However, we do not know yet whether TERT actually protects mtDNA against oxidative damage, whether it activates repair of mtDNA or whether it accelerates degradation of mitochondria with damaged DNA. It is also not clear yet whether the improved maintenance of mtDNA is a cause or a consequence of the lower levels of ROS production in TERT-expressing fibroblasts. However, it is important to note that a relatively mild mitochondrial dysfunction as induced here by an increase in ambient oxygen concentration might occur under a wide variety of physiological or mildly pathological situations. Thus, we propose a telomere-independent function for telomerase, namely a protective role against mtDNA damage by mitochondrially derived ROS. The fact that essentially all of TERT is transported out of the nucleus under long-term low level oxidative stress raises the issue of whether protection of mitochondrial DNA might be of more immediate consequence than protection of telomeres under stress.
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Materials and Methods |
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-H2A.X foci, and flow cytometric analysis of cells stained with MitoSOX, dihydrorhodamine 123, JC-1 or nonyl Acridine Orange (NAO, all Molecular Probes) were performed as described previously (Passos et al., 2007). To measure apoptosis, the activity of the `executioner caspases' 3 and 7 was assessed using Apo-one homogeneous caspase-3/7 assay (Promega).
siRNAs A and B against TERT as described (Shammas et al., 2005) and control siRNA (AM4611, Ambion) were transfected using siPORT Amine (Ambion) at a concentration of 30 nM. TERT expression was measured by RT-PCR using primers 5'-GCCTGAGCTGTACTTTGTCAA-3' (forward) and 5'-GCAAACAGCTTGTTTCTCCATGTC-3' (reverse).
Gene expression
Total RNA was extracted from the following independent cultures using RNAeasy (Qiagen): four mid-passage parental MRC5, three last passage MRC5 (senescent) and three MRC5-TERT, all grown under normoxia. All cultures were confluency-arrested in G0. Biotin-labelled cRNA was prepared and processed according to the manufacturer's instructions and analysed using U133 2.0 plus gene chips (Affymetrix, Buckingham, UK). Data were analysed using GeneSpring v7.0 using the following criteria: at least three out of 10 calls marginal or present, average fold change between conditions at least twofold.
Telomere length and telomerase activity
Telomere length was measured by in gel hybridisation and Telomere Q-FISH on metaphases as described (Passos et al., 2007; von Zglinicki et al., 2000). Telomerase activity was analysed with a TeloTAGGG Telomerase PCR ELISA kit (Roche). Signal linearity was established over an input range of 0.1 to 20 ng of protein, and measurements were performed with 2 and 10 ng protein per sample. A DNA/Cytosol fractionation kit (Bio Vision) was used to separate nuclei. A crude mitochondrial fraction was isolated as described (Stuart et al., 2004). Purity of fractions was tested by western blotting using antibodies against HDAC2 (Abcam) and COX2 (SantaCruz).
mtDNA damage and copy number
Relative mtDNA copy number was determined as amplification efficiency of an 83 bp mtDNA amplicon using real-time PCR as described (Passos et al., 2007); however, the heat denaturation step preceding the PCR reaction was prolonged to 6 minutes to allow full denaturation of supercoiled mtDNA.
mtDNA damage was measured by two independent methods. First, we used long-range PCR (Santos et al., 2006a) to measure the amplification efficiency for a large (11,095bp) mtDNA fragment in real-time PCR as described (Passos et al., 2007). Second, we assessed damage by its effect on the supercoiled state of mtDNA. After 2 minutes of heat denaturation at 94°C, intact mtDNA remains largely in a supercoiled state (being resistant to amplification by PCR), while damaged mtDNA is relaxed and can be amplified. After 6 minutes at 94°C, all mtDNA is relaxed and can be amplified (Chen et al., 2007). Thus, relative mtDNA damage was measured as the ratio of amplification efficiencies for the 83 bp amplicon [primer nucleotide sequences 5'-GATTTGGGTACCACCCAAGTATTG-3' (16042-16064) and 5'-AATATTCATGGTGGCTGGCATGTA-3' (16125-16102)] following 2 and 6 minutes of heat denaturation. Both amplifications were performed using SYBR Green JumpStart Taq ReadyMix (Sigma) in an MJ Chromo4. Cycle conditions following heat denaturation were: 30 seconds at 94°C, 45 seconds at 60°C, 45 seconds at 72°C.
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Acknowledgments |
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Footnotes |
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References |
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Armstrong, L., Saretzki, G., Peters, H., Wappler, I., Evans, J., Hole, N., von Zglinicki, T. and Lako, M. (2005). Overexpression of telomerase confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic lineage. Stem Cells 23, 516-529.
Bagheri, S., Nosrati, M., Li, S., Fong, S., Torabian, S., Rangel, J., Moore, D. H., Federman, S., Laposa, R. R., Baehner, F. L. et al. (2006). Genes and pathways downstream of telomerase in melanoma metastasis. Proc. Natl. Acad. Sci. USA 103, 11306-11311.
Bakalova, R., Ohba, H., Zhelev, Z., Ishikawa, M., Shinohara, Y. and Baba, Y. (2003). Cross-talk between Bcr-Abl tyrosine kinase, protein kinase C and telomerase-a potential reason for resistance to Glivec in chronic myelogenous leukaemia. Biochem. Pharmacol. 66, 1879-1884.[CrossRef][Medline]
Balaban, R. S., Nemoto, S. and Finkel, T. (2005). Mitochondria, oxidants, and aging. Cell 120, 483-495.[CrossRef][Medline]
Biswas, G., Guha, M. and Avadhani, N. G. (2005). Mitochondria-to-nucleus stress signaling in mammalian cells: nature of nuclear gene targets, transcription regulation, and induced resistance to apoptosis. Gene 354, 132-139.[CrossRef][Medline]
Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S. and Wright, W. E. (1998). Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349-352.
Butow, R. A. and Avadhani, N. G. (2004). Mitochondrial signaling: the retrograde response. Mol. Cell 14, 1-15.[CrossRef][Medline]
Chen, J., Kadlubar, F. F. and Chen, J. Z. (2007). DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair. Nucleic Acids Res. 35, 1377-1388.
d'Adda di Fagagna, F., Reaper, P. M., Clay-Farrace, L., Fiegler, H., Carr, P., Von Zglinicki, T., Saretzki, G., Carter, N. P. and Jackson, S. P. (2003). A DNA damage checkpoint response in telomere-initiated senescence. Nature 426, 194-198.[CrossRef][Medline]
del Bufalo, D., Rizzo, A., Trisciuoglio, D., Cardinali, G., Torrisi, M. R., Zangemeister-Wittke, U. and Biroccio, A. (2005). Involvement of hTERT in apoptosis induced by interference with Bcl-2 expression and function. Cell Death Differ. 12, 1429-1438.[CrossRef][Medline]
Dimri, G. P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E. E., Linskens, M., Rubelj, I., Pereira-Smith, O. et al. (1995). A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92, 9363-9367.
Dudognon, C., Pendino, F., Hillion, J., Saumet, A., Lanotte, M. and Segal-Bendirdjian, E. (2004). Death receptor signaling regulatory function for telomerase: hTERT abolishes TRAIL-induced apoptosis, independently of telomere maintenance. Oncogene 23, 7469-7474.[CrossRef][Medline]
Franco, S., Canela, A., Klatt, P. and Blasco, M. A. (2005). Effectors of mammalian telomere dysfunction: a comparative transcriptome analysis using mouse models. Carcinogenesis 26, 1613-1626.
Fu, W., Killen, M., Culmsee, C., Dhar, S., Pandita, T. K. and Mattson, M. P. (2000). The catalytic subunit of telomerase is expressed in developing brain neurons and serves a cell survival-promoting function. J. Mol. Neurosci. 14, 3-15.[CrossRef][Medline]
Furumoto, K., Inoue, E., Nagao, N., Hiyama, E. and Miwa, N. (1998). Age-dependent telomere shortening is slowed down by enrichment of intracellular vitamin C via suppression of oxidative stress. Life Sci. 63, 935-948.[CrossRef][Medline]
Geserick, C., Tejera, A., Gonzalez-Suarez, E., Klatt, P. and Blasco, M. A. (2006). Expression of mTert in primary murine cells links the growth-promoting effects of telomerase to transforming growth factor-beta signaling. Oncogene 25, 4310-4319.[CrossRef][Medline]
Gonzalez-Suarez, E., Goytisolo, F. A., Flores, J. M. and Blasco, M. A. (2003). Telomere dysfunction results in enhanced organismal sensitivity to the alkylating agent N-methyl-N-nitrosourea. Cancer Res. 63, 7047-7050.
Haendeler, J., Hoffmann, J., Brandes, R. P., Zeiher, A. M. and Dimmeler, S. (2003). Hydrogen peroxide triggers nuclear export of telomerase reverse transcriptase via Src kinase family-dependent phosphorylation of tyrosine 707. Mol. Cell. Biol. 23, 4598-4610.
Haendeler, J., Hoffmann, J., Diehl, J. F., Vasa, M., Spyridopoulos, I., Zeiher, A. M. and Dimmeler, S. (2004). Antioxidants inhibit nuclear export of telomerase reverse transcriptase and delay replicative senescence of endothelial cells. Circ. Res. 94, 768-775.
Jazwinski, S. M. (2005). The retrograde response links metabolism with stress responses, chromatin-dependent gene activation, and genome stability in yeast aging. Gene 354, 22-27.[CrossRef][Medline]
Kang, H. J., Choi, Y. S., Hong, S. B., Kim, K. W., Woo, R. S., Won, S. J., Kim, E. J., Jeon, H. K., Jo, S. Y., Kim, T. K. et al. (2004). Ectopic expression of the catalytic subunit of telomerase protects against brain injury resulting from ischemia and NMDA-induced neurotoxicity. J. Neurosci. 24, 1280-1287.
Kondo, Y., Kondo, S., Tanaka, Y., Haqqi, T., Barna, B. P. and Cowell, J. K. (1998). Inhibition of telomerase increases the susceptibility of human malignant glioblastoma cells to cisplatin-induced apoptosis. Oncogene 16, 2243-2248.[CrossRef][Medline]
Kurz, D. J., Decary, S., Hong, Y., Trivier, E., Akhmedov, A. and Erusalimsky, J. D. (2004). Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells. J. Cell Sci. 117, 2417-2426.
Lee, K. H., Rudolph, K. L., Ju, Y. J., Greenberg, R. A., Cannizarro, L., Chin, L., Weiler, S. R. and DePinho, R. A. (2001). Telomere dysfunction alters the chemotherapeutic profile of transformed cells. Proc. Natl. Acad. Sci. USA 98, 3381-3386.
Li, S., Crothers, J., Haqq, C. M. and Blackburn, E. H. (2005). Cellular and gene expression responses involved in the rapid growth inhibition of human cancer cells by RNA interference-mediated depletion of telomerase RNA. J. Biol. Chem. 280, 23709-23717.
Liu, K., Hodes, R. J. and Weng, N. (2001). Cutting edge: telomerase activation in human T lymphocytes does not require increase in telomerase reverse transcriptase (hTERT) protein but is associated with hTERT phosphorylation and nuclear translocation. J. Immunol. 166, 4826-4830.
Ludwig, A., Saretzki, G., Holm, P. S., Tiemann, F., Lorenz, M., Emrich, T., Harley, C. B. and von Zglinicki, T. (2001). Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase. Cancer Res. 61, 3053-3061.
Martin-Ruiz, C., Saretzki, G., Petrie, J., Ladhoff, J., Jeyapalan, J., Wei, W., Sedivy, J. and von Zglinicki, T. (2004). Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span. J. Biol. Chem. 279, 17826-17833.
Massard, C., Zermati, Y., Pauleau, A. L., Larochette, N., Metivier, D., Sabatier, L., Kroemer, G. and Soria, J. C. (2006). hTERT: A novel endogeneous inhibitor of the mitochondrial cell death pathway. Oncogene 25, 4505-4514.[CrossRef][Medline]
Nautiyal, S., DeRisi, J. L. and Blackburn, E. H. (2002). The genome-wide expression response to telomerase deletion in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 99, 9316-9321.
Oh, H., Taffet, G. E., Youker, K. A., Entman, M. L., Overbeek, P. A., Michael, L. H. and Schneider, M. D. (2001). Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival. Proc. Natl. Acad. Sci. USA 98, 10308-10313.
Passos, J., Saretzki, G., Ahmed, S., Nelson, G., Richter, T., Peters, H., Wappler, I., Birket, M., Harold, G., Schaeuble, K. et al. (2007). Mitochondrial dysfunction accounts for the stochastic heterogeneity in telomere-dependent senescence. PLoS Biol. 5, e110.[CrossRef][Medline]
Petersen, S., Saretzki, G. and von Zglinicki, T. (1998). Preferential accumulation of single-stranded regions in telomeres of human fibroblasts. Exp. Cell Res. 239, 152-160.[CrossRef][Medline]
Santos, J. H., Meyer, J. N., Skorvaga, M., Annab, L. A. and Van Houten, B. (2004). Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage. Aging Cell 3, 399-411.[CrossRef][Medline]
Santos, J. H., Meyer, J. N., Mandavilli, B. S. and Van Houten, B. (2006a). Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol. Biol. 314, 183-199.[Medline]
Santos, J. H., Meyer, J. N. and Van Houten, B. (2006b). Mitochondrial localization of telomerase as a determinant for hydrogen peroxide-induced mitochondrial DNA damage and apoptosis. Hum. Mol. Genet. 15, 1757-1768.
Sarin, K. Y., Cheung, P., Gilison, D., Lee, E., Tennen, R. I., Wang, E., Artandi, M. K., Oro, A. E. and Artandi, S. E. (2005). Conditional telomerase induction causes proliferation of hair follicle stem cells. Nature 436, 1048-1052.[CrossRef][Medline]
Shammas, M. A., Koley, H., Batchu, R. B., Bertheau, R. C., Protopopov, A., Munshi, N. C. and Goyal, R. K. (2005). Telomerase inhibition by siRNA causes senescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeutic potential. Mol. Cancer 4, 1-14.[CrossRef][Medline]
Sharma, G. G., Gupta, A., Wang, H., Scherthan, H., Dhar, S., Gandhi, V., Iliakis, G., Shay, J. W., Young, C. S. and Pandita, T. K. (2003). hTERT associates with human telomeres and enhances genomic stability and DNA repair. Oncogene 22, 131-146.[CrossRef][Medline]
Shay, J. W. and Bacchetti, S. (1997). A survey of telomerase activity in human cancer. Eur. J. Cancer 33, 787-791.[CrossRef][Medline]
Smith, L. L., Coller, H. A. and Roberts, J. M. (2003). Telomerase modulates expression of growth-controlling genes and enhances cell proliferation. Nat. Cell Biol. 5, 474-479.[CrossRef][Medline]
Stewart, S. A., Hahn, W. C., O'Connor, B. F., Banner, E. N., Lundberg, A. S., Modha, P., Mizuno, H., Brooks, M. W., Fleming, M., Zimonjic, D. B. et al. (2002). Telomerase contributes to tumorigenesis by a telomere length-independent mechanism. Proc. Natl. Acad. Sci. USA 99, 12606-12611.
Stuart, J. A., Hashiguchi, K., Wilson, D. M., Copeland, W. C., Souza-Pinto, N. C. and Bohr, V. A. (2004). DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA. Nucleic Acids Res. 32, 2181-2192.
von Zglinicki, T. (2002). Oxidative stress shortens telomeres. Trends Biochem. Sci. 27, 339-344.[CrossRef][Medline]
von Zglinicki, T., Pilger, R. and Sitte, N. (2000). Accumulation of single stranded breaks is the major cause of telomere shortening in human fibroblasts. Free Radic. Biol. Med. 28, 64-74.[CrossRef][Medline]
von Zglinicki, T., Saretzki, G., Ladhoff, J., d'Adda di Fagagna, F. and Jackson, S. P. (2005). Human cell senescence as a DNA damage response. Mech. Ageing Dev. 126, 111-117.[CrossRef][Medline]
Wu, Y. L., Dudognon, C., Nguyen, E., Hillion, J., Pendino, F., Tarkanyi, I., Aradi, J., Lanotte, M., Tong, J. H., Chen, G. Q. et al. (2006). Immunodetection of human telomerase reverse-transcriptase (hTERT) re-appraised: nucleolin and telomerase cross paths. J. Cell Sci. 119, 2797-2806.
Zhang, P., Chan, S. L., Fu, W., Mendoza, M. and Mattson, M. P. (2003). TERT suppresses apoptotis at a premitochondrial step by a mechanism requiring reverse transcriptase activity and 14-3-3 protein-binding ability. FASEB J. 17, 767-769.
Zhu, H., Fu, W. and Mattson, M. P. (2000). The catalytic subunit of telomerase protects neurons against amyloid beta-peptide-induced apoptosis. J. Neurochem. 75, 117-124.[CrossRef][Medline]
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HINDIII size marker. (B) Telomere shortening rates per PD in MRC5 (black bars) and MRC5-TERT (white bars) under normoxia (left) and hyperoxia (right). Data are mean±s.e.m. from four experiments measured in quadruplicate. The asterisk indicates a significant difference between parental and TERT-overexpressing cells with P<0.05 (ANOVA). (C) Metaphase spreads from MRC5 (left) and MRC5-TERT (right) cells grown under hyperoxia for the indicated times (in days) hybridised with a telomeric PNA probe (red). Chromosomes are stained with DAPI (blue). (D) Frequency distributions of telomere fluorescence signal intensities in MRC5-TERT (top) and MRC5 (bottom) cells grown under hyperoxia for the indicated times. Red bars indicate the median telomere length. (E) Frequencies of chromosomal end-to-end fusions per metaphase in MRC5 (black) and MRC5-TERT (white, broken lines) under hyperoxia. Data are mean±s.e.m. from at least 25 metaphases per condition.
