7015

TERT

telomerase reverse transcriptase;TERT;telomerase reverse transcriptase

telomerase catalytic subunit|telomerase-associated protein 2

5p15.33

protein-coding

cells subjected to sub-lethal doses of stress such as irradiation or oxidative damage enter a state that closely resembles replicative senescence. What triggers stress-induced premature senescence (SIPS) and how similar this mechanism is to replicative senescence are not well understood. It has been suggested that stress-induced senescence is caused by rapid telomere shortening resulting from DNA damage. In order to test this hypothesis directly, we examined whether overexpression of the catalytic subunit of human telomerase (hTERT) can protect cells from SIPS. We therefore analyzed the response of four different lines of normal human fibroblasts with and without hTERT to stress induced by UV, gamma-irradiation, and H(2)O(2). SIPS was induced with the same efficiency in normal and hTERT-immortalized cells. This suggests that SIPS is not triggered by telomere shortening and that nonspecific DNA damage serves as a signal for induction of SIPS. Although telomerase did not protect cells from SIPS, fibroblasts expressing hTERT were more resistant to stress-induced apoptosis and necrosis. We hypothesize that healing of DNA breaks by telomerase inhibits the induction of cell death, but because healing does not provide legitimate DNA repair, it does not protect cells from SIPS.

Reconstitution of telomerase activity by ectopic expression of telomerase reverse transcriptase (hTERT) results in an immortal phenotype in various types of normal human cells, including fibroblasts. Despite lack of transformation characteristics, it is unclear whether hTERT-immortalized cells are physiologically and biochemically the same as their normal counterparts. Here, we compared the gene expression profiles of normal and hTERT-immortalized fibroblasts by using a cDNA microarray containing 20,736 cDNA clones and identified 172 dysregulated genes or expressed sequence tags (ESTs). One of the highly expressed genes in the hTERT-immortalized fibroblasts (hTERT-BJ cells) encodes epiregulin, a potent growth factor. Blockade of epiregulin reduced the growth of hTERT-BJ cells and colony formation of hTERT-transformed fibroblasts. Moreover, inhibition of epiregulin function in immortal hTERT-BJ cells triggered a senescence program. Our results suggest that both activation of telomerase and subsequent induction of epiregulin are required for sustained cell proliferation. Given the significant difference in gene expression profiles between normal and hTERT-immortalized fibroblasts and the close relationship between epiregulin and tumorigenesis, we conclude that hTERT-immortalized cells may not replace their normal counterparts for studies of normal cell biology and that the use of hTERT for expansion of normal human cells for therapeutic purposes must be approached with caution.

Little is known about the long-term consequences of overexpression of the human telomerase reverse transcriptase (hTERT) gene in T lymphocytes. To address this issue, we transduced polyclonal as well as clonally derived populations of naive and memory CD44 T cells from 2 healthy donors (aged 24 and 34 years) with retroviral vectors encoding green fluorescence protein (GFP) and hTERT (GFP-hTERT) or GFP alone. After transduction, cells were sorted on the basis of GFP expression and cultured in vitro until senescence. T cells transduced with hTERT exhibited high stable telomerase activity throughout the culture period. Relative to GFP controls, minor changes in overall gene expression were observed yet the proliferative lifespan of the hTERT-transduced populations was significantly increased and the rate of telomere loss was lower. Nevertheless, hTERT-transduced cells showed progressive telomere loss and had shorter telomeres at senescence than controls (2.3 +/- 0.3 kilobase [kb] versus 3.4 +/- 0.1 kb). Furthermore, a population of cells with 4N DNA consisting of binucleated cells with connected nuclei emerged in the hTERT-transduced cells prior to senescence. We conclude that overexpression of hTERT in CD4+ T cells provides a proliferative advantage independent of the average telomere length but does not prevent eventual genetic instability and replicative senescence.

BACKGROUND: Recent studies have demonstrated that aging or senescence constitutes a potential limitation to the ability of endothelial progenitor cells (EPCs) to sustain ischemic tissue and repair. Conversely, estrogens have been shown to accelerate recovery of the endothelium after vascular injury. OBJECTIVE: To investigate whether estrogens are able to prevent senescence of EPCs. METHODS AND RESULTS: Human EPCs were isolated from peripheral blood and characterized. After ex-vivo cultivation, the cells became senescent as determined by acidic beta-galactosidase staining. 17beta-estradiol dose-dependently inhibited the onset of EPC senescence in culture. Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity using a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique. 17beta-estradiol significantly increased telomerase activity. Interestingly, reverse transcriptase-PCR analysis demonstrated that 17beta-estradiol dose-dependently increased the catalytic subunit, telomerase reverse transcriptase (TERT) - an effect that was significantly inhibited by pharmacological phosphatidylinositol 3-kinase (PI3-K) blockers (either wortmannin or LY294002). Because the expression of TERT is regulated by the PI3-K/Akt pathway, we examined the effect of 17beta-estradiol on Akt activity in EPCs. Immunoblotting analysis revealed that 17beta-estradiol dose-dependently led to phosphorylation and, thus, to activation of Akt in EPCs. We also examined whether the protective effect of 17beta-estradiol on EPC senescence translates into the augmentation of mitogenic activity in EPCs. A [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tet razolium] (MTS) assay demonstrated that the mitogenic potential in EPCs treated with 17beta-estradiol exceeded that in untreated (control) EPCs (P < 0.01). In addition, EPCs released vascular endothelial growth factor (VEGF) protein--an effect that was significantly augmented by 17beta-estradiol. Finally, in a Matrigel assay, EPCs treated with both 17beta-estradiol and VEGF were shown to be more likely to integrate into the network formation than those treated with VEGF alone. CONCLUSION: The inhibition of EPC senescence by estrogen in vitro may improve the functional activity of EPCs in a way that is important for potential cell therapy.

Even though meningiomas are the second most common brain tumor in adults, little is known about the molecular basis of their growth and development. The lack of suitable cell culture model systems is an impediment to this understanding. Most studies on meningiomas rely on primary, early passage cell lines that eventually senesce or a few established cell lines that have been derived from aggressive variants of meningiomas. We have isolated three primary meningioma cell lines that are negative for telomerase activity. We can overcome the senescence of a Grade III derived meningioma cell line by expressing the telomerase catalytic subunit (hTERT), whereas Grade I meningioma cell lines require the expression of the human papillomavirus E6 and E7 oncogenes in conjunction with hTERT. Meningioma cell lines, immortalized in this manner, maintain their pre-transfection morphology and form colonies in vitro. We have confirmed the meningothelial origin of these cell lines by assessing expression of vimentin and desmoplakin, characteristic markers for meningiomas. Additionally, we have karyotyped these cell lines using array CGH and shown that they represent a spectrum of the genetic diversity seen in primary meningiomas. Thus, these cell lines represent novel cellular reagents for investigating the molecular oncogenesis of meningiomas.

Activation of telomerase is sufficient for immortalization of some types of human cells but additional factors may also be essential. It has been proposed that stress imposed by inadequate culture conditions induces senescence due to accumulation of p16(INK4a). Here, we present evidence that many human cell types undergo senescence by activation of the p16(INK4a)/Rb pathway, and that introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span of human epithelial cells derived from skin, mammary gland and lung. Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1, suppressed p16(INK4a) expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT, the telomerase catalytic subunit, into cells with low p16(INK4a) levels resulted in efficient immortalization of three cell types without crisis or growth arrest. The majority of the human mammary epithelial cells thus immortalized showed almost normal ploidy as judged by G-banding and spectral karyotyping analysis. Our data suggest that inhibition of p16(INK4a) and introduction of hTERT can immortalize many human cell types with little chromosomal instability.

Fibroblasts derived from glucose-6-phosphate dehydrogenase (G6PD)-deficient patients display retarded growth and accelerated cellular senescence that is attributable to increased accumulation of oxidative DNA damage and increased sensitivity to oxidant-induced senescence, but not to accelerated telomere attrition. Here, we show that ectopic expression of hTERT stimulates telomerase activity and prevents accelerated senescence in G6PD-deficient cells. Stable clones derived from hTERT-expressing normal and G6PD-deficient fibroblasts have normal karyotypes, and display no sign of senescence beyond 145 and 105 passages, respectively. Activation of telomerase, however, does not prevent telomere attrition in earlier-passage cells, but does stabilize telomere lengths at later passages. In addition, we provide evidence that ectopic expression of hTERT attenuates the increased sensitivity of G6PD-deficient fibroblasts to oxidant-induced senescence. These results suggest that ectopic expression of hTERT, in addition to acting in telomere length maintenance by activating telomerase, also functions in regulating senescence induction.

Recent studies have suggested that reduced endothelial progenitor subpopulation in lectin-binding and DiLDL-uptaking cell (EPC subpopulation) number and activity was associated with EPC subpopulation senescence that involved telomerase activity and telomere length. Stromal cell-derived factor-1alpha (SDF-1alpha) has been shown to augment a variety of cellular functions of EPC subpopulation and subsequently contribute to ischemic neovascularization. Therefore, we investigated whether SDF-1alpha might be able to prevent senescence of EPC subpopulation and also investigated the effects of SDF-1alpha on the telomerase activity and telomere length. EPC subpopulation were isolated from peripheral blood and characterized. After ex vivo prolonged cultivation, EPC subpopulation became senescent as determined by acidic beta-galactosidase staining. SDF-1alpha dose-dependently inhibited the onset of EPC subpopulation senescence. Moreover, SDF-1alpha increased proliferation and colony-forming activity of EPC subpopulation. SDF-1alpha also increased telomerase activity and telomere length, which was accompanied with upregulation of the catalytic subunit, telomerase reverse transcriptase (TERT). Whereas these effects of SDF-1alpha on telomerase activity and expression of hTERT mRNA were significantly attenuated by CXCR4-specific peptide antagonist (AMD3100) and phosphoinositide 3-kinase (PI3K) inhibitor (LY294002). In conclusions, SDF-1alpha delays the onset of EPC subpopulation senescence, which may be related to the activation of telomerase and elongation of telomere length. The inhibition of EPC subpopulation senescence and induction of EPC subpopulation proliferation by SDF-1alpha in vitro may importantly improve the functional activity of EPC subpopulation for potential cell therapy.

BACKGROUND: Telomerase is a ribonucleoprotein enzyme capable of extending chromosome ends with telomeric DNA sequences. It protects the germline and stem cells from senescence by preventing telomere attrition. Cutaneous aging includes intrinsic aging, and photo-aging. Telomere-associated cellular senescence contributes to certain age-related cutaneous disorders, including increased cancer incidence. Premature skin aging in xeroderma pigmentosa (XP) is expected to show increased telomere attrition. We aimed to study human telomerase reverse transcriptase (hTERT) expression in normal, aged and photo-aged skin and to investigate its possible role in the pathogenesis of aging and photo-aging. METHODS: hTERT expression using immunohistochemistry was studied in 75 subjects comprising four groups: group I, 10 subjects with aged skin; group II, 20 subjects with photo-aging; group III, Five patients with XP; and group IV, 40 subjects comprising the control groups. RESULTS: We found positive hTERT in normal skin and in the basal and sometimes in supra-basal layers. We reported positive hTERT expression in dermal fibroblasts, histiocytes, and skin appendages (other than hair follicles) in some cases from ALL the studied groups. Photo-aged and prematurely photo-aged skin showed greater hTERT expression than young and aged skin. CONCLUSION: Telomeres rather than telomerase are involved in cellular senescence. Yet, telomerase is intimately related to photo-aging in which lifetime cumulative sun exposure is an important factor. However, genetic damage in XP is the decisive factor and not merely ultraviolet exposure.

OBJECTIVE: Telomeres and associated proteins are regulators of cellular survival, regeneration and aging. PPAR-gamma agonists may mediate vascular effects in addition to insulin sensitizing. We therefore examined whether pioglitazone regulates vascular telomere biology. METHODS AND RESULTS: C57/Bl6 mice were randomized to treatment with pioglitazone (20mg/kg i.p. daily) or vehicle for 4 weeks (n = 6-8 per group). Telomere repeat amplification protocols showed a 2-fold increase of aortic telomerase activity in the pioglitazone group. Telomere repeat-binding factor 2 protein and mRNA levels (236%+172% of vehicle) as well as phosphorylation of protein kinase Akt (479% of vehicle) were up-regulated. Western blots demonstrated reduced aortic expression of senescence markers p16, cell-cycle checkpoint kinase 2 and p53. These regulatory mechanisms were independent of acute changes of telomere length. Similar observations were made in mononuclear cells (MNC) from these mice and in cultivated bovine aortic endothelial cells, human MNC and endothelial progenitor cells (EPC). Telomerase activation by pioglitazone in cultivated cells was prevented by Akt inhibitors. To test the functional relevance of the findings, isolated mononuclear cells (MNC) were exposed to H(2)O(2). MNC from pioglitazone-treated mice exhibited reduced apoptosis (AnnexinV-FACS). In vivo, lipopolysaccharide-induced aortic endothelial apoptosis was potently prevented in pioglitazone-treated animals (hairpin oligonucleotide assay). Both, up-regulation of telomere-regulating proteins and prevention of oxidative stress-induced aortic apoptosis, were absent in telomerase reverse transcriptase (TERT)-deficient mice. CONCLUSIONS/INTERPRETATION: Pioglitazone treatment up-regulates telomerase activity, telomere-stabilizing proteins and reduces senescence markers in vascular cells. These effects and the reduction of LPS-induced endothelial apoptosis by thiazolidinediones depend on TERT. The findings underscore the important role of telomere-regulating proteins for vascular cell function and survival.