hTERT Promoter Regulation by Differentiation Mechanisms vs Telomerase Activity in Somatic, Embryonic, and Cancerous Cells

OBJECTIVEChemotherapeutic options for meningiomas refractory to surgery or irradiation are largely unknown. Human telomerase reverse transcriptase (hTERT) promoter methylation with subsequent TERT expression and telomerase activity, key features in oncogenesis, are found in most high-grade meningiomas. Therefore, the authors investigated the impact of the demethylating agent decitabine (5-aza-2ʹ-deoxycytidine) on survival and DNA methylation in meningioma cells.METHODShTERT promoter methylation, telomerase activity, TERT expression, and cell viability and proliferation were investigated prior to and after incubation with decitabine in two benign (HBL-52 and Ben-Men 1) and one malignant (IOMM-Lee) meningioma cell line. The global effects of decitabine on DNA methylation were additionally explored with DNA methylation profiling.RESULTSHigh levels of TERT expression, telomerase activity, and hTERT promoter methylation were found in IOMM-Lee and Ben-Men 1 but not in HBL-52 cells. Decitabine induced a dose-dependent significant decrease of proliferation and viability after incubation with doses from 1 to 10 μM in IOMM-Lee but not in HBL-52 or Ben-Men 1 cells. However, effects in IOMM-Lee cells were not related to TERT expression, telomerase activity, or hTERT promoter methylation. Genome-wide methylation analyses revealed distinct demethylation of 14 DNA regions after drug administration in the decitabine-sensitive IOMM-Lee but not in the decitabine-resistant HBL-52 cells. Differentially methylated regions covered promoter regions of 11 genes, including several oncogenes and tumor suppressor genes that to the authors’ knowledge have not yet been described in meningiomas.CONCLUSIONSDecitabine decreases proliferation and viability in high-grade but not in benign meningioma cell lines. The effects of decitabine are TERT independent but related to DNA methylation changes of promoters of distinct tumor suppressor genes and oncogenes.

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CpG island methylation of the hTERT promoter is associated with lower telomerase activity in B-cell lymphocytic leukemia

Experimental Hematology ◽

10.1016/s0301-472x(01)00760-3 ◽

2002 ◽

Vol 30 (1) ◽

pp. 26-33 ◽

Cited By ~ 48

Author(s):

O Bechter

Keyword(s):

B Cell ◽

Cpg Island ◽

Telomerase Activity ◽

Lymphocytic Leukemia ◽

Htert Promoter ◽

Cpg Island Methylation

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Epidermal growth factor activates telomerase activity by direct binding of Ets-2 to hTERT promoter in lung cancer cells

Tumor Biology ◽

10.1007/s13277-015-3204-x ◽

2015 ◽

Vol 36 (7) ◽

pp. 5389-5398 ◽

Cited By ~ 6

Author(s):

Chung-Ping Hsu ◽

Li-Wen Lee ◽

Sheau-Chung Tang ◽

I-Lun Hsin ◽

Yu-Wen Lin ◽

...

Keyword(s):

Lung Cancer ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Cancer Cells ◽

Telomerase Activity ◽

Lung Cancer Cells ◽

Htert Promoter ◽

Direct Binding ◽

Epidermal Growth

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Central role of PI3K in transcriptional activation of hTERT in HTLV-I–infected cells

Blood ◽

10.1182/blood-2008-01-134692 ◽

2008 ◽

Vol 112 (7) ◽

pp. 2946-2955 ◽

Cited By ~ 34

Author(s):

Marcia Bellon ◽

Christophe Nicot

Keyword(s):

T Cell ◽

Wilms Tumor ◽

Telomerase Activity ◽

Pi3k Pathway ◽

T Cell Leukemia ◽

Htert Promoter ◽

Cell Leukemia ◽

Human T Cell ◽

Infected Cells ◽

I Cells

Abstract The persistence of human T-cell leukemia/lymphoma virus-I (HTLV-I)–infected cells is dependent upon clonal expansion and up-regulation of telomerase (hTERT). We have previously found that in interleukin (IL)–2–independent transformed HTLV-I cells, Tax strongly activates the hTERT promoter through nuclear factor-κB (NF-κB)–mediated Sp1 and c-Myc activation. In IL-2–dependent cells and adult T-cell leukemia/lymphoma (ATLL) patient samples, however, Tax expression is very low to undetectable, yet these cells retain strong telomerase activity. This suggests the existence of compensatory mechanisms in IL-2–dependent cells and ATLL patients. In this study, we demonstrate that telomerase activity is significantly decreased upon IL-2 withdrawal in immortalized HTLV-I cell lines. Inhibition of PI3K or AKT signaling pathways reduced telomerase activity in HTLV-I cells. We found that IL-2/IL-2R signaling was associated with a PI3K-dependent/AKT-independent transcriptional up-regulation of the endogenous hTERT promoter. We found that activation of the PI3K pathway mediated cytoplasmic retention of the Wilms tumor (WTI) protein, which strongly suppressed the hTERT promoter. The importance of this regulatory pathway for telomerase expression is underscored by findings that the PI3K pathway is commonly found activated in cancer cells.

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Latent Membrane Protein 1 of Epstein-Barr Virus Activates the hTERT Promoter and Enhances Telomerase Activity in B Lymphocytes

Journal of Virology ◽

10.1128/jvi.00321-08 ◽

2008 ◽

Vol 82 (20) ◽

pp. 10175-10187 ◽

Cited By ~ 46

Author(s):

Liliana Terrin ◽

Jessica Dal Col ◽

Enrica Rampazzo ◽

Paola Zancai ◽

Moreno Pedrotti ◽

...

Keyword(s):

B Cells ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

Ectopic Expression ◽

Telomerase Activity ◽

Latent Membrane Protein ◽

Latent Membrane Protein 1 ◽

Htert Promoter ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Transformation of primary B lymphocytes by Epstein-Barr virus requires the establishment of a strictly latent infection, the expression of several latent viral proteins, and sustained telomerase activity. Our previous findings indicated that induction of hTERT, the rate-limiting catalytic unit of the telomerase complex, was associated with the expression of the viral latent membrane protein 1 (LMP1). In the present study, we demonstrate that ectopic expression of LMP1 in BJAB and Ramos B cells resulted in an increase of hTERT transcripts, thus suggesting that LMP1 acts at the transcriptional level. This was confirmed by transient expression of a luciferase reporter plasmid containing the hTERT promoter cotransfected with an LMP1-expressing vector or transfected into B cells in which LMP1 expression was inducible. Consistently, silencing of LMP1 by small interfering RNA resulted in a reduction of hTERT transcripts. We also provide evidence indicating that LMP1-induced hTERT activation is independently mediated by NF-κB and by mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways, whereas CD40, Akt, and mTOR signaling has no involvement. Moreover, our results do not support a role for c-Myc in mediating these effects on hTERT, since ectopic expression of LMP1 did not upregulate c-Myc and silencing of this oncogene or E box mutagenesis failed to inhibit LMP1-induced hTERT activation. These findings indicate that LMP1 simultaneously modulates multiple signal transduction pathways in B cells to transactivate the hTERT promoter and enhance telomerase activity, thus confirming the pleiotropic nature of this viral oncoprotein.

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Hypoxia-Inducible Factor 1 Mediates Upregulation of Telomerase (hTERT)

Molecular and Cellular Biology ◽

10.1128/mcb.24.13.6076-6083.2004 ◽

2004 ◽

Vol 24 (13) ◽

pp. 6076-6083 ◽

Cited By ~ 128

Author(s):

Hirotaka Nishi ◽

Toshihide Nakada ◽

Satoru Kyo ◽

Masaki Inoue ◽

Jerry W. Shay ◽

...

Keyword(s):

Promoter Activity ◽

Hypoxia Inducible Factor ◽

Major Complication ◽

Cancer Therapeutics ◽

First Trimester ◽

Telomerase Activity ◽

Htert Promoter ◽

Htert Expression ◽

Human Disorders

ABSTRACT Hypoxia occurs during the development of the placenta in the first trimester and correlates with both trophoblast differentiation and the induction of telomerase activity through hTERT expression. We sought to determine the mechanism of regulation of hTERT expression during hypoxia. We show that hypoxia-inducible factor 1α (HIF-1α) and hTERT expression in the human placenta decrease with gestational age and that these are overexpressed in preeclamptic placenta, a major complication of pregnancy. Hypoxia not only transactivates the hTERT promoter activity but also enhances endogenous hTERT expression. The hTERT promoter region between −165 and +51 contains two HIF-1 consensus motifs, and in vitro reporter assays show that these are essential for hTERT transactivation by HIF-1. Introduction of an antisense oligonucleotide for HIF-1 diminishes hTERT expression during hypoxia, indicating that upregulation of hTERT by hypoxia is directly mediated through HIF-1. Our results provide persuasive evidence that the regulation of hTERT promoter activity by HIF-1 represents a mechanism for trophoblast growth during hypoxia and suggests that this may be a generalized response to hypoxia in various human disorders including resistance to cancer therapeutics by upregulating telomerase.

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A DNA tetrahedron docking assembly for imaging telomerase activity in cancerous cells

Analytica Chimica Acta ◽

10.1016/j.aca.2021.339395 ◽

2021 ◽

pp. 339395

Author(s):

Ruixue Zhang ◽

Ruiyuan Zhang ◽

Chunyan Zhao ◽

Xiaowen Xu

Keyword(s):

Telomerase Activity ◽

Dna Tetrahedron ◽

Cancerous Cells

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Development of Adenoviral Vectors for Detection of Proliferation and Differentiation Stage Dependent Telomerase Reverse Transcriptase Expression in Single Living Hematopoietic Cells.

Blood ◽

10.1182/blood.v104.11.4413.4413 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4413-4413

Author(s):

Xiaolong Fan ◽

Anna Edqvist ◽

Johan Rebetz ◽

Marcus Nilsson-Jaras ◽

Leif G. Salford ◽

...

Keyword(s):

Cell Cycle ◽

Hematopoietic Cells ◽

Telomerase Activity ◽

Adenoviral Vectors ◽

Leukemic Cells ◽

Telomerase Reverse Transcriptase ◽

Htert Promoter ◽

Proliferation And Differentiation ◽

Single Living ◽

Insulator Sequence

Abstract Elevated telomerase activity is an important molecular signature for proliferating hematopoietic progenitors, lymphocytes and a variety of leukemic cells. The human telomerase activity is predominantly determined by the transcriptional control of the catalytic subunit telomerase reverse transcriptase (hTERT). For prospective identification of single living hematopoietic cells with telomerase activity, we have developed telomerase-reporting adenoviral vectors encoding destabilized enhanced green fluorescence protein with a half-life of 2 hours (d2EGFP) driven by the hTERT promoter. To shield the hTERT promoter from adenoviral backbone cis-acting DNA sequences, vectors encoding the chicken β-globin locus HS4 insulator sequence or bovine growth hormone transcription stop signal were also engineered. Moreover, these vectors were retargeted with the Ad35 fiber receptor specificity (Ad5F35 vectors) and utilize ubiquitously expressed CD46 as a cellular receptor. The Ad5F35 vectors allow efficient gene delivery into hematopoietic cells (J Gene Med6:631, 2004). The telomerase activity dependent d2EGFP expression was demonstrated in telomerase positive HeLa, A549 or negative fibroblasts and WI-38 cells by infection with the telomerase-reporting vectors at 2000 physical particles per cells. Successful gene delivery was achieved as about 90% of the cells were EGFP+ following infection with control Ad5F35-CMV-EGFP vector. Between 40 to 95% of the individual HeLa and A549 cells were d2EGFP+, and the percentages of the d2EGFP+ cells positively correlated with the extent of the telomerase activity as assessed in Trap assay. In contrast, a barely detectable fraction of the WI-38 cells and lower than 6% of the fibroblasts were expressing lower levels of d2EGFP. The vector carrying the HS4 insulator sequence generated the lowest background d2EGFP expression in fibroblasts and WI-38 cells (called cTERTdGFP vector). Therefore the cTERTdGFP vector was used for all the subsequent studies. Importantly, no measurable d2EGFP expression was observed in normal primary myeloid cells. The cTERTdGFP vector mediated d2EGFP expression in hematopoietic cells is dynamically regulated during cell proliferation and differentiation. Promyelocytic leukemic HL-60 cells were infected with the cTERTdGFP or the control Ad5F35-PGK-EGFP vector. Nearly 100% of the HL-60 cells were EGFP+ following the control vector infection, however, around 50% of the HL-60 cells were d2EGFP+ following cTERTdGFP vector infection. The d2EGFP expression was reduced 2-fold with lower d2EGFP intensity in retinoic acid induced differentiating HL-60 cells compared with non-treated HL-60 cells. HL-60 cells expanded from sorted d2EGFP+ or d2EGFP- cells generated identical frequencies of d2EGFP+ and d2EGFP- cells following re-infection with the cTERTdGFP vector. Cell cycle analysis assessing cellular DNA content in combination with Ki-67 antigen staining or BrdU pulsing showed that the sorted d2EGFP+ HL-60 cells contained significantly fewer cells in the G1, more cells in the S/G2/M phase of cell cycle compared with the d2EGFP- HL-60 cells. In conclusion, our studies can provide a powerful tool for isolation of living normal hematopoietic or leukemic cells with telomerase activity from heterogeneous cell populations.

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Cell-Restricted Immortalization by Human Papillomavirus Correlates with Telomerase Activation and Engagement of the hTERT Promoter by Myc

Journal of Virology ◽

10.1128/jvi.01318-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11568-11576 ◽

Cited By ~ 37

Author(s):

Xuefeng Liu ◽

Aleksandra Dakic ◽

Renxiang Chen ◽

Gary L. Disbrow ◽

Yiyu Zhang ◽

...

Keyword(s):

High Risk ◽

Core Promoter ◽

Human Fibroblasts ◽

Human Keratinocytes ◽

Telomerase Activity ◽

Human Papillomaviruses ◽

Htert Promoter ◽

Causative Agents ◽

E6 And E7 ◽

Interfering Rna

ABSTRACT The high-risk human papillomaviruses (HPVs) are the causative agents of nearly all cervical cancers and are etiologically linked to additional human cancers, including those of anal, oral, and laryngeal origin. The main transforming genes of the high-risk HPVs are E6 and E7. E6, in addition to its role in p53 degradation, induces hTERT mRNA transcription in genital keratinocytes via interactions with Myc protein, thereby increasing cellular telomerase activity. While the HPV type 16 E6 and E7 genes efficiently immortalize human keratinocytes, they appear to only prolong the life span of human fibroblasts. To examine the molecular basis for this cell-type dependency, we examined the correlation between the ability of E6 to transactivate endogenous and exogenous hTERT promoters and to immortalize genital keratinocytes and fibroblasts. Confirming earlier studies, the E6 and E7 genes were incapable of immortalizing human fibroblasts but did delay senescence. Despite the lack of immortalization, E6 was functional in the fibroblasts, mediating p53 degradation and strongly transactivating an exogenous hTERT promoter. However, E6 failed to transactivate the endogenous hTERT promoter. Coordinately with this failure, we observed that Myc protein was not associated with the endogenous hTERT promoter, most likely due to the extremely low level of Myc expression in these cells and/or to differences in chromatin structure, in contrast with hTERT promoters that we found to be activated by E6 (i.e., the endogenous hTERT promoter in primary keratinoctyes and the exogenous hTERT core promoter in fibroblasts), where Myc is associated with the promoter in either a quiescent or an E6-induced state. These findings are consistent with those of our previous studies on mutagenesis and the knockdown of small interfering RNA, which demonstrated a requirement for Myc in the induction of the hTERT promoter by E6 and suggested that occupancy of the promoter by Myc determines the responsiveness of E6 and the downstream induction of telomerase and cell immortalization.

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Hierarchical Organization of Acute Myeloid Leukemia Characterized by Promoter Activity of the Human Telomerase Reverse Transcriptase Gene.

Blood ◽

10.1182/blood.v104.11.3381.3381 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3381-3381

Author(s):

Seiichiro Kobayashi ◽

Yasushi Soda ◽

Yuansong Bai ◽

Hiroyuki Miyoshi ◽

Shigetaka Asano ◽

...

Keyword(s):

Cell Population ◽

Promoter Activity ◽

Leukemia Cell ◽

Viable Cell ◽

Telomerase Activity ◽

Hierarchical Organization ◽

Leukemia Cells ◽

Htert Promoter ◽

Human Telomerase Reverse Transcriptase ◽

Cd34 Cells

Abstract Acute myeloid leukemia (AML) is generally believed to originate from hematopoietic stem cells and to constitute a hierarchy of heterogeneous populations in terms of proliferation. To gain insight into the hierarchical organization of AML, a lentiviral reporter assay, based on the expression of novel yellow fluorescent protein variant (Venus) using flow cytometry, was applied to the promoter activity of the human telomerase reverse transcriptase (hTERT) gene in primary AML cells. hTERT promoter activity correlated well with endogenous telomerase activity in experiments inducing apoptosis and differentiation of leukemia cell lines. Next, AML cells or purified cord blood (CB) CD34+ cells transduced with hTERT-Venus (the lentiviral vector containing the expression cassette of the Venus gene driven by the hTERT promoter) were cultured in the presence or absence of colony-stimulating factors (CSF) mixtures (SCF, IL3, GM-CSF, and G-CSF) for 48 hrs before analysis. In primary AML cases, hTERT promoter activity revealed considerable patient-to-patient variation, regardless of stimulation by CSF, but its distribution appeared as a single peak or two narrowly separated peaks, suggesting that hTERT expression occurs along a continuous gradient in the leukemia cell population. The mean promoter activity of the 19 AML cells was 2-fold higher than that of CB CD34+ cells without CSF, but almost equal to that with CSF. In four cases, the MFI was not responsive to CSF stimulation, and in three cases, the basal and stimulated MFI were prominent. In contrast, three lots of CB CD34+ cells uniformly revealed very low activity (MFI) without stimulation (mean=1.2), but 5-fold up-regulation with stimulation (mean=5.8). Without CSF stimulation, hTERT promoter activity was slightly higher in CD34+CD38− cells than in CD34+CD38+ cells in most AML cases, and leukemia cells in S/G2/M phase had greater promoter activity than those in G0/G1 phase. Next, to monitor the changes in hTERT promoter activity during short-term culture, hTERT-Venus transduced leukemia cells were maintained with CSF mixtures, and Venus expression was analyzed using flow cytometry at Days 2, 6, and 10. A representative case is shown in the Figure. Both the viable cell population and its hTERT promoter activity increased at Day 6. The viable cell population then decreased, but the fluorescence intensity of this fraction still increased markedly at Day 10. This might be due to the gradual disappearance of the cell fraction with lower hTERT promoter activity, which suggests that leukemia cells with higher telomerase activity survive in a mass population. Furthermore, AML cells expected to have high telomerase activity could be identified under fluorescent microscopy, making it possible to track their behavior in culture. In conclusion, we presented here novel evidence for a hierarchy of telomerase activity, deduced from hTERT promoter activity, among individual leukemia cells in AML. Figure Figure

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