Transcriptional Activation Of hTERT Research Articles

MAPK/ERK-CBP-RFPL-3 Mediates Adipose-Derived Stem Cell-Induced Tumor Growth in Breast Cancer Cells by Activating Telomerase Reverse Transcriptase Expression.

Adipose-derived stem cells (ASCs) improve the self-renewal and survival of fat grafts in breast reconstruction after oncology surgery. However, ASCs have also been found to enhance breast cancer growth, and its role in tumor proliferation remains largely elusive. Here, we explored a novel mechanism that mediates hTERT reactivation during ASC-induced tumor growth in breast cancer cells. In this study, we found the proliferative ability of breast cancer cells markedly increased with ASC coculture. To explore the molecular mechanism, we treated cells with anibody/inhibitor and found that the activation of MEK-ERK pathway was triggered in breast cancer cells by SCF secreted from ASCs, leading to the nuclear recruitment of CBP. As a coactivator of hTERT, CBP subsequently coordinated with RFPL-3 upregulated hTERT transcription and telomerase activity. The inhibition of CBP and RFPL-3 abrogated the activation of hTERT transcription and the promotion of proliferation in breast cancer cells with cocultured ASCs in vitro and in vivo. Collectively, our study findings indicated that CBP coordination with RFPL-3 promotes ASC-induced breast cancer cell proliferation by anchoring to the hTERT promoter and upregulating telomerase activity, which is activated by the MAPK/ERK pathway.

Functional role of SETD2, BAP1, PARP-3 and PBRM1 candidate genes on the regulation of hTERT gene expression.

Narrowing the search for the critical hTERT repressor sequence(s) has identified three regions on chromosome 3p (3p12-p21.1, 3p21.2 and 3p21.3-p22). However, the precise location and identity of the sequence(s) responsible for hTERT transcriptional repression remains elusive. In order to identify critical hTERT repressor sequences located within human chromosome 3p12-p22, we investigated hTERT transcriptional activity within 21NT microcell hybrid clones containing chromosome 3 fragments. Mapping of chromosome 3 structure in a single hTERT-repressed 21NT-#3fragment hybrid clone, revealed a 490kb region of deletion localised to 3p21.3 and encompassing the histone H3, lysine 36 (H3K36) trimethyltransferase enzyme SETD2; a putative tumour suppressor gene in breast cancer. Three additional genes, BAP1, PARP-3 and PBRM1, were also selected for further investigation based on their location within the 3p21.1-p21.3 region, together with their documented role in the epigenetic regulation of target gene expression or hTERT regulation. All four genes (SETD2, BAP1, PARP-3 and PBRM1) were found to be expressed at low levels in 21NT. Gene copy number variation (CNV) analysis of SETD2, BAP1, PARP-3 and PBRM1 within a panel of nine breast cancer cell lines demonstrated single copy number loss of all candidate genes within five (56%) cell lines (including 21NT cells). Stable, forced overexpression of BAP1, but not PARP2, SETD2 or PBRM1, within 21NT cells was associated with a significant reduction in hTERT expression levels relative to wild-type controls. We propose that at least two sequences exist on human chromosome 3p, that function to regulate hTERT transcription within human breast cancer cells.

Baculovirus Vector-Mediated Transfer of Sodium Iodide Symporter and Plasminogen Kringle 5 Genes for Tumor Radioiodide Therapy

BackgroundBoth tumor cells and their supporting endothelial cells should be considered for targeted cell killing when designing cancer treatments. Here we investigated the feasibility of combining radioiodide and antiangiogenic therapies after baculovirus-mediated transfer of genes encoding the sodium iodide symporter (NIS) and plasminogen kringle 5 (K5).MethodsA recombinant baculovirus containing the NIS gene under control of the human telomerase reverse transcriptase (hTERT) promoter and the K5 gene driven by the early growth response 1 (Egr1) promoter was developed. Dual-luciferase reporter assay was performed to confirm the activation of hTERT transcription. NIS and K5 gene expression were identified by Western blot and Real-Time PCR. Functional NIS activity in baculovirus-infected Hela cells was confirmed by the uptake of 125I and cytotoxicity of 131I. The apoptotic effect of 131I-induced K5 on baculovirus-infected human umbilical vein endothelial cells (HUVECs) was analyzed by a flow cytometry-based assay. In vivo, NIS reporter gene imaging and therapeutic experiments with 131I were performed. Finally, the microvessel density (MVD) in tumors after treatment was determined by CD31 immunostaining.ResultsThe activation of hTERT transcription was specifically up-regulated in tumor cells. NIS gene expression markedly increased in baculovirus-infected HeLa cells, but not in MRC5 cells. The Hela cells showed a significant increase of 125I uptake, which was inhibited by NaClO4, and a notably decreased cell survival rate by 131I treatment. Expression of the K5 gene induced by 131I was elevated in a dose- and time-dependent manner and resulted in the apoptosis of HUVECs. Furthermore, 131I SPECT imaging clearly showed cervical tumor xenografts infected with recombinant baculovirus. Following therapy, tumor growth was significantly retarded. CD31 immunostaining confirmed a significant decrease of MVD.ConclusionThe recombinant baculovirus supports a promising strategy of NIS-based raidoiodide therapy combined with K5-based antiangiogenic therapy by targeting both the tumor and its supporting vessels.

Enrichment of human osteosarcoma stem cells based on hTERT transcriptional activity.

Telomerase is crucial for the maintenance of stem/progenitor cells in adult tissues and is detected in most malignant cancers, including osteosarcoma. However, the relationship between telomerase expression and cancer stem cells remains unknown. We observed that sphere-derived osteosarcoma cells had higher telomerase activity, indicating that telomerase activity might be enriched in osteosarcoma stem cells. We sorted subpopulations with high or low telomerase activity (TEL) using hTERT transcriptional promoter-induced green fluorescent protein (GFP). The TELpos cells showed an increased sphere and tumor propagating capacity compared to TELneg cells, and enhanced stem cell-like properties such as invasiveness, metastatic activity and resistance to chemotherapeutic agents both in vitro and in vivo. Furthermore, the telomerase inhibitor MST312 prevented tumorigenic potential both in vitro and in vivo, preferentially targeting the TELpos cells. These data support telomerase inhibition as a potential targeted therapy for osteosarcoma stem-like cells.

HTLV-1 bZIP factor impedes the menin tumor suppressor and upregulates JunD-mediated transcription of the hTERT gene

Telomerase activity in cancer cells is dependent on the transcriptional regulation of the human telomerase reverse transcriptase (hTERT) gene, encoding the catalytic subunit of human telomerase. We have shown previously that HTLV-1 basic leucine zipper (HBZ), a viral regulatory protein encoded by the human retrovirus, human T-cell leukemia virus, type 1 (HTLV-1) cooperates with JunD to enhance hTERT transcription in adult T-cell leukemia (ATL) cells. Menin, the product of the tumor-suppressor MEN-1 gene, also interacts with JunD, represses its transcriptional activity and downregulates telomerase expression. The main objective of this study was to examine how menin and HBZ get involved in the regulation of hTERT transcription. In this study, we report that JunD and menin form a repressor complex of hTERT transcription in HBZ-negative cells. Conversely, in HBZ-positive cells, the formation of a JunD/HBZ/menin ternary complex and the recruitment of p300 histone acetyl transferase activity by HBZ lead to a decreased activity of the JunD-menin suppressor unit that correlates with the activation of hTERT transcription. Silencing HBZ or menin expression in ATL cells confirms that these proteins are differentially involved in telomerase regulation. These results propose that HBZ, by impeding the tumor-suppressor activity of menin, functions as a leukemogenic cofactor to upregulate gene transcription and promote JunD-mediated leukemogenesis.

Aspirin inhibits human telomerase activation in unstable carotid plaques

The activation of telomerase in unstable plaques is an important factor in atherosclerosis, and may be predictive of the risk of cerebrovascular diseases. Human telomerase reverse transcriptase (hTERT) is a subunit of telomerase that is essential for telomerase activation. The aim of the present study was to investigate whether aspirin inhibits the activation of telomerase and hTERT in unstable carotid plaques. Polymorphonuclear neutrophils (PMNs) derived from carotid plaques were isolated from the washing medium of angioplasty balloons, while circulating PMNs, isolated from arterial blood, served as the controls. A polymerase chain reaction-based telomeric repeat amplification protocol (TRAP) enzyme-linked immunosorbent assay (ELISA) was used to measure the telomerase activity in the cells following treatment with aspirin. The mRNA and protein expression of hTERT were detected by a reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The results revealed that the atherosclerotic plaques were positive for telomerase activity, and that aspirin inhibited the telomerase activity of the PMNs derived from the plaques. In addition, aspirin was demonstrated to inhibit the mRNA and protein expression of hTERT through the suppression of hTERT transcriptional activity; however, it had no inhibitory effect on the telomerase activity of the circulating PMNs. Thus, the activation of telomerase in resident PMNs is critical in the instability of carotid plaques. The upregulation of telomerase and hTERT during the progression of atherosclerosis may indicate a role for telomerase in the vascular remodeling that occurs during atherogenesis. Aspirin was demonstrated to inhibit the activation of telomerase via an hTERT-dependent manner in the PMN cells of unstable carotid plaques, and thus hTERT may be considered as a target in the treatment of cerebrovascular diseases.

Use of the Comet-FISH Assay to Compare DNA Damage and Repair in p53 and hTERT Genes following Ionizing Radiation

The alkaline single cell gel electrophoresis (comet) assay can be combined with fluorescent in situ hybridisation (FISH) methodology in order to investigate the localisation of specific gene domains within an individual cell. The number and position of the fluorescent signal(s) provides information about the relative damage and subsequent repair that is occurring in the targeted gene domain(s). In this study, we have optimised the comet-FISH assay to detect and compare DNA damage and repair in the p53 and hTERT gene regions of bladder cancer cell-lines RT4 and RT112, normal fibroblasts and Cockayne Syndrome (CS) fibroblasts following γ-radiation. Cells were exposed to 5Gy γ-radiation and repair followed for up to 60 minutes. At each repair time-point, the number and location of p53 and hTERT hybridisation spots was recorded in addition to standard comet measurements. In bladder cancer cell-lines and normal fibroblasts, the p53 gene region was found to be rapidly repaired relative to the hTERT gene region and the overall genome, a phenomenon that appeared to be independent of hTERT transcriptional activity. However, in the CS fibroblasts, which are defective in transcription coupled repair (TCR), this rapid repair of the p53 gene region was not observed when compared to both the hTERT gene region and the overall genome, proving the assay can detect variations in DNA repair in the same gene. In conclusion, we propose that the comet-FISH assay is a sensitive and rapid method for detecting differences in DNA damage and repair between different gene regions in individual cells in response to radiation. We suggest this increases its potential for measuring radiosensitivity in cells and may therefore have value in a clinical setting.

Butylidenephthalide Suppresses Human Telomerase Reverse Transcriptase (TERT) in Human Glioblastomas

Telomerase is widely expressed in most human cancers, but is almost undetectable in normal somatic cells and is therefore a potential drug target. Using the human telomerase promoter platform, the naturally occurring compound butylidenephthalide (BP) was selected for subsequent investigation of antitumor activity in vitro and in vivo. We treated human glioblastoma cells with BP and found a dose-dependent decrease in human telomerase reverse transcriptase (hTERT) mRNA expression and a concomitant increase in p16 and p21 expression. Because c-Myc and Sp1 are involved in transcriptional regulation of hTERT, the effect of BP on c-Myc and Sp1 expression was examined. Using electrophoretic mobility shift assays and western blotting, we showed that BP represses hTERT transcriptional activity via downregulation of Sp1 expression. Using the telomerase repeat amplification protocol, an association between BP concentration and suppression of telomerase activity, induction of human glioblastoma senescence, and inhibition of cellular proliferation was identified. This was supported by a mouse xenograft model, in which BP repressed telomerase and inhibited tumor proliferation, resulting in tumor senescence. Overexpression of hTERT restored telomerase activity in human glioblastoma cells and overcame replicative senescence. These findings suggest that BP inhibits proliferation and induces senescence in human glioblastomas by downregulating hTERT expression and consequently telomerase activity. This is the first study to describe regulation of telomerase activity by BP in human glioblastomas.

Identification and Characterization of Bmi-1-responding Element within the Human p16 Promoter*

Bmi-1, the first functionally identified polycomb gene family member, plays critical roles in cell cycle regulation, cell immortalization, and cell senescence. Bmi-1 is involved in the development and progression of carcinomas and is a potent target for cancer therapy. One important pathway regulated by Bmi-1 is that involving two cyclin-dependent kinase inhibitors, p16(Ink4a) and p19(Arf), as Bmi-1 represses the INK4a locus on which they are encoded. A close correlation between the up-regulation of Bmi-1 and down-regulation of p16 has been demonstrated in various tumors; however, how Bmi-1 regulates p16 expression is not clear. In this study, we revealed that Bmi-1 regulates the expression of p16 by binding directly to the Bmi-1-responding element (BRE) within the p16 promoter. The BRE resided at bp -821 to -732 upstream of the p16 ATG codon. BRE alone was sufficient to allow Bmi-1-mediated regulation of the CMV promoter. Bmi-1 typically functions by forming a complex with Ring2; however, regulation of p16 was independent of Ring2. Chromatin immunoprecipitation sequencing of Bmi-1-precipitated chromatin DNA revealed that 1536 genes were targeted by Bmi-1, including genes involved in tissue-specific differentiation, cell cycle, and apoptosis. By analyzing the binding sequences of these genes, we found two highly conserved Bmi-1-binding motifs, which were required for Bmi-1-mediated p16 promoter regulation. Taken together, our results revealed the molecular mechanism of Bmi-1-mediated regulation of the p16 gene, thus providing further insights into the functions of Bmi-1 as well as a sensitive high-throughput platform with which to screen Bmi-1-targeted small molecules for cancer therapy.

Aberrant Control of Transcription and Transportation of Oncogenic mRNA in Carcinogenesis

Multiple gene disorders have been shown to be involved in carcinogenesis. Mutation, translocation and amplification have been identified in so-called oncogenes, and inactivation of antioncogenes by mutation and deletion has been shown. E1AF is an ets-oncogene family transcription factor, and has been shown to upregulate multiple matrix metalloproteinase (MMP) genes that contribute to the malignant phenotype of cancer cells by inducing invasive and metastatic activities. EWS/ETS fusions are frequently observed in Ewing's sarcoma, and we have revealed that EWS/ETS chimeric protein activates telomerase activity by upregulating human telomerase reverse transcriptase (hTERT), but the transcriptional activation of hTERT by EWS/ETS was indirect, and EWS/ETS was seen to function as a co-activator for TERT transcription. A number of oncogenes and cancer-related genes contain AU-rich element (ARE) in non-coding regions of transcribed mRNA. HuR is a RNA-binding protein that has the potential to stabilize ARE-containing mRNAs. HuR is known to shuttle between the nucleus and the cytoplasm via several export pathways. When normal cells are exposed to stress, HuR is exported to the cytoplasm in a chromosome maintenance region 1 (CRM1)-dependent manner. However, we demonstrate that HuR is CRM-1 independently exported to the cytoplasm in oral cancer cells. ARE-mRNAs were also exported to the cytoplasm and stabilized in the oral cancer cells, which were inhibited by HuR knockdown. These findings suggest that transcriptional and translational abnormalities of oncogenes may contribute to the carcinogenesis of oral epithelial cells.

Abstract 3204: The mRNA decay factor tristetraprolin (TTP) induces senescence in human papillomavirus-transformed cervical cancer cells by targeting E6-AP ubiquitin ligase

Abstract Cervical cancer is the second most common cancer among women worldwide. A necessary factor in the development of nearly all cases of cervical cancer is the human papillomavirus (HPV) infection. High-risk HPV (HPV16 and 18) mediates cellular transformation and carcinogenesis through the concerted actions of viral oncoproteins E6 and E7 that disrupt the normal cell cycle regulation by targeting the p53 and Rb tumor suppressor pathways respectively. In the present study, we found that expression of the mRNA decay factor TTP is lost in HPV18-positive HeLa cells and introduction of TTP in cells lead to inhibition of cell proliferation and tumorigenesis by inducing cellular senescence. Consistent with this, TTP-expressing cells showed enhanced p53 protein stability and p53-dependent transcriptional activity. In cells expressing TTP, no changes were observed in the RNA levels of E6 and E7 indicating that these viral factors are not targets of TTP-mediated RNA decay. Whereas the E6-associated protein (E6-AP), the cellular ubiquitin ligase required for E6-mediated p53 degradation, was found to be down regulated both at mRNA and protein levels in presence of TTP. Furthermore, the association of E6 and E6-AP promotes transcriptional activation of hTERT, and TTP-dependent down regulation of E6-AP resulted in inhibition of hTERT expression and decreased cellular telomerase activity. Analysis of E6-AP mRNA revealed potential AU-rich element (ARE) binding sites of TTP present in the 3’ untranslated region (3′UTR) and RNA-binding studies demonstrate a physical interaction between TTP and E6-AP mRNA leading to rapid decay of E6-AP mRNA in a 3’UTR-dependent manner. Similar results were obtained with other high-risk HPV-positive cell lines that employ E6-AP pathway to control p53 and hTERT levels. Consistent with this, we found TTP expression to be lost or significantly reduced in squamous cell carcinomas as compared to normal cervical tissue. Finally, our studies indicate that loss of TTP expression in cervical cancer occurs through epigenetic mechanisms involving promoter methylation and histone deacetylation. These findings demonstrate the ability of TTP to act as a tumor suppressor through inhibition of the E6-AP pathway and indicate TTP loss to be a critical event during HPV-mediated carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3204.

Telomerase upregulation is a postcrisis event during senescence bypass and immortalization of two Nijmegen breakage syndrome T cell cultures

Our knowledge on immortalization and telomere biology is mainly based on genetically manipulated cells analyzed before and many population doublings post growth crisis. The general view is that growth crisis is telomere length (TL) dependent and that escape from crisis is coupled to increased expression of the telomerase reverse transcriptase (hTERT) gene, telomerase activity upregulation and TL stabilization. Here we have analyzed the process of spontaneous immortalization of human T cells, regarding pathways involved in senescence and telomerase regulation. Two Nijmegen breakage syndrome (NBS) T cell cultures (S3R and S4) showed gradual telomere attrition until a period of growth crisis followed by the outgrowth of immortalized cells. Whole genome expression analysis indicated differences between pre-, early post- and late postcrisis cells. Early postcrisis cells demonstrated a logarithmic growth curve, very short telomeres and, notably, no increase in hTERT or telomerase activity despite downregulation of several negative hTERT regulators (e.g. FOS, JUN D, SMAD3, RUNX2, TNF-a and TGFb-R2). Thereafter, cMYC mRNA increased in parallel with increased hTERT expression, telomerase activity and elongation of short telomeres, indicating a step-wise activation of hTERT transcription involving reduction of negative regulators followed by activation of positive regulator(s). Gene expression analysis indicated that cells escaped growth crisis by deregulated DNA damage response and senescence controlling genes, including downregulation of ATM, CDKN1B (p27), CDKN2D (p19) and ASF1A and upregulation of CDK4, TWIST1, TP73L (p63) and SYK. Telomerase upregulation was thus found to be uncoupled to escape of growth crisis but rather a later event in the immortalization process of NBS T cell cultures.

PAX5 activates the transcription of the human telomerase reverse transcriptase gene in B cells

Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. Its activity is not detectable in most somatic cells but it is reactivated during tumorigenesis. In most cancers, the combination of hTERT hypermethylation and hypomethylation of a short promoter region is permissive for low-level hTERT transcription. Activated and malignant lymphocytes express high telomerase activity, through a mechanism that seems methylation-independent. The aim of this study was to determine which mechanism is involved in the enhanced expression of hTERT in lymphoid cells. Our data confirm that in B cells, some T cell lymphomas and non-neoplastic lymph nodes, the hTERT promoter is unmethylated. Binding sites for the B cell-specific transcription factor PAX5 were identified downstream of the ATG translational start site through EMSA and ChIP experiments. ChIP assays indicated that the transcriptional activation of hTERT by PAX5 does not involve repression of CTCF binding. In a B cell lymphoma cell line, siRNA-induced knockdown of PAX5 expression repressed hTERT transcription. Moreover, ectopic expression of PAX5 in a telomerase-negative normal fibroblast cell line was found to be sufficient to activate hTERT expression. These data show that activation of hTERT in telomerase-positive B cells is due to a methylation-independent mechanism in which PAX5 plays an important role.

Hepatitis B virus protein preS2 potentially promotes HCC development via its transcriptional activation of hTERT

Backgrounds and aims:Telomerase is significantly reactivated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC). Our previous studies showed that the transactivation unit of HBV surface (S) gene, preS2, could upregulate...

Induction of apoptosis and inhibition of telomerase activity in human lung carcinoma cells by the water extract of Cordyceps militaris

Cordyceps militaris is well known as a traditional medicinal mushroom and is a potentially interesting candidate for use in cancer treatment. In this study, the potential of the water extract of C. militaris (WECM) to induce apoptosis in human lung carcinoma A549 cells and its effects on telomerase activity were investigated. The growth inhibition and apoptosis induction by WECM treatment in A549 cells was associated with the induction of Fas, catalytic activation of caspase-8, and Bid cleavage. Activation of caspases, downregulation of anti-apoptotic Bcl-2 expression, and upregulation of pro-apoptotic Bax protein were also observed in WECM-treated cells. However, the cytotoxic effects and apoptotic characteristics induced by WECM were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 plays in the process. In addition, WECM exerted a dose-dependent inhibition of telomerase activity via downregulation of human telomerase reverse transcriptase (hTERT), c-myc and Sp1 expression. Taken together, the data from this study indicate that WECM induces the apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic and mitochondria-mediated intrinsic caspase pathways. It was also conclude that apoptotic events due to WECM were mediated with diminished telomerase activity through the inhibition of hTERT transcriptional activity.

NFX1-123 Increases hTERT Expression and Telomerase Activity Posttranscriptionally in Human Papillomavirus Type 16 E6 Keratinocytes

High-risk human papillomavirus (HPV) E6 protein induces telomerase activity through transcriptional activation of hTERT, the catalytic subunit of telomerase. HPV type 16 (HPV16) E6 interacts with two splice variants of NFX1 to increase hTERT expression. NFX1-91 is a transcriptional repressor of hTERT that is polyubiquitinated and targeted for degradation by HPV16 E6 in concert with E6-associated protein. We previously showed that NFX1-123 augments hTERT expression through binding to cytoplasmic poly(A) binding proteins (PABPCs). In this study, we determined that unlike NFX1-91, NFX1-123 is a cytoplasmic protein that colocalized with PABPCs but does not shuttle with PABPCs between the nucleus and cytoplasm. NFX1-123 requires both its PAM2 motif, with which it binds PABPCs, and its R3H domain, which has putative nucleic acid binding capabilities, to increase hTERT mRNA levels and telomerase activity in keratinocytes expressing HPV16 E6. In keratinocytes expressing HPV16 E6 and overexpressing NFX1-123, there was increased protein expression from in vitro-transcribed RNA fused with the 5' untranslated region (5' UTR) of hTERT. This posttranscriptional increase in expression required the PAM2 motif and R3H domain of NFX1-123 as well as the coexpression of HPV16 E6. NFX1-123 bound endogenous hTERT mRNA and increased its stability in HPV16 E6-expressing human foreskin keratinocytes, and NFX1-123 increased the stability of in vitro-transcribed RNA fused with the 5' UTR of hTERT. Together, these studies describe the first evidence of posttranscriptional regulation of hTERT, through the direct interaction of the cytoplasmic protein NFX1-123 with hTERT mRNA, in HPV16 E6-expressing keratinocytes.

Human Telomerase Reverse Transcriptase Activated by E6 Oncoprotein Is Required for Human Papillomavirus-16/18-Infected Lung Tumorigenesis

Our recent report indicates that human papillomavirus (HPV)-16/18 E6 oncoprotein is expressed in lung tumors and is related to p53 inactivation. We further explored whether human telomerase reverse transcriptase (hTERT) transcription is up-regulated by E6 and contributes to lung tumor development. Immunohistochemistry detected HPV-16 E6 oncoprotein in 135 lung tumors, and hTERT mRNA was evaluated by real-time reverse transcription-PCR and in situ hybridization, respectively. A small RNA interference (RNAi), Western blotting, and chromatin immunoprecipitation analysis were used to clarify whether hTERT transcription was regulated by c-Myc and Sp1. The telomerase activity and oncogenic potential of TL-1 with or without E6- or hTERT-RNAi was determined by real-time quantitative telomeric repeat amplification protocol analysis and soft-agar assay, respectively. hTERT mRNA levels in E6-positive tumors, which were prevalent in females, nonsmokers, and adenocarcinomas, were significantly higher than in E6-negative tumors. In addition, hTERT mRNA levels in early tumors (stage I) were greater than levels in advanced tumors (stages II and III). Chromatin immunoprecipitation assay showed that Sp1 cooperated with c-Myc to activate hTERT transcription in TL-1 cells, which was similar to the SiHa cells. The telomerase activity of the TL-1 cells decreased concomitantly with the transfection of various doses of E6- or hTERT-RNAi. A soft-agar assay showed that the oncogenic potential of TL-1 cells was significantly reduced after being transfected with E6-RNAi. Moreover, a colony of TL-1 cells could not form after transfection with hTERT-RNAi. Transcriptional activation of hTERT by E6 oncoprotein is required for HPV-16/18-infected lung tumorigenesis.

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

AbstractThe 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.

Early growth response‐1 mediates downregulation of telomerase in cervical cancer

Early growth response (Egr)-1 is a transcription factor that triggers transcription of downstream genes within 15-30 min of various stimulations. These genes are expressed rapidly through specific promoter activation and mediate cell growth and angiogenesis. Following the previous computational identification of a site that was thought to be an Egr-1 consensus binding site at -273 to -281 in the human telomerase reverse transcriptase (hTERT) promoter region, the present study was conducted to evaluate the role of Egr-1 in the regulation of hTERT and telomerase in uterine cervical cancer. First, the expression of Egr-1 and hTERT at the mRNA level was examined in cervical cancer tissues. Egr-1 and hTERT were expressed much higher in cervical cancer tissues than in the normal cervix. However, a negative correlation was noted in the expression between Egr-1 and hTERT. By luciferase assay using hTERT promoter constructs, hTERT transcriptional activation was shown to be inhibited when Egr-1 was overexpressed. Furthermore, Egr-1 overexpression decreased hTERT protein production as well as hTERT mRNA as observed by western blotting analysis and real-time reverse transcription-polymerase chain reaction, respectively. The present study suggests that Egr-1 plays an important regulatory role in the transcriptional activation of hTERT.

Posttranscriptional regulation of the telomerase hTERT by gambogic acid in human gastric carcinoma 823 cells

We previously reported that gambogic acid (GA), a natural product, was an effective telomerase inhibitor by repressing hTERT promoter. In this study, posttranscriptional regulation of the telomerase hTERT by GA was investigated in BGC-823 human gastric carcinoma cells. The telomerase activity was detected by PCR-TRAP assay. RT-PCR assay and Western blot were performed to examine the repression of telomerase hTERT and c-Myc after GA or c-Myc-specific siRNA treatment. The results indicated that GA repressed telomerase activity and hTERT transcriptional activity via down-regulation of c-Myc expression in BGC-823 human gastric carcinoma cells. We further observed that hTERT transcriptional activity was not completely blocked by c-Myc-specific siRNA, suggesting that additional factors are involved in the repression of telomerase activity. The results of Western blot and immunoprecipitation assay revealed that GA inhibits the phosphorylation of Akt. The further results also confirmed that celecoxib, an inhibitor of Akt phosphorylation, could significantly repressed telomerase activity alone and enhance the repression of telomerase activity combined with GA. These data suggested that GA inhibits the posttranslational modification of hTERT by inhibiting the phosphorylation of Akt. Collectively, we suggest that GA represses telomerase activity not only by repressing hTERT transcriptional activity via c-Myc but also by posttranslational modification of hTERT via Akt.