hTERT mRNA Levels Research Articles

Influence of the hTERT rs2736100 polymorphism on telomere length in gastric cancer.

To investigate the functional consequences of rs2736100 polymorphism in telomere length and examine its link to gastric cancer risk. Telomere length and human telomerase reverse transcriptase (hTERT) mRNA expression were measured in 35 gastric cancer tissues and 5 cell lines and correlated to rs2736100 polymorphism. The relationship between rs2736100 polymorphism and the risk of gastric cancer were examined in 243 gastric cancer patients and 246 healthy individuals. The rs2736100 A allele carrier is closely associated with reduced hTERT mRNA expression and shortened telomere length in gastric cancer tissue and cell lines. When gastric cancers were stratified by histological subtype, telomere length and hTERT mRNA levels were significantly increased in those with the C/C genotype in intestinal-type gastric cancer, but not in diffuse-type gastric cancer. Interestingly, there was no significant difference in the genotype and allele frequencies of the rs2736100 polymorphism between the patients with gastric cancer and healthy controls. The rs2736100 polymorphism of the hTERT gene is involved in the regulation of hTERT expression and telomere length, but not in the risk of gastric cancer.

PTEN and hTERT gene expression and the correlation with human hepatocellular carcinoma

The aim of this study was to investigate the correlation between tumor suppressor gene phosphatase and tensin homolog (PTEN) expression levels and telomerase activity that mainly depends on telomerase reverse transcriptase (hTERT) in hepatocellular carcinoma (HCC) and paracancerous tissues. Immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of PTEN and hTERT in 58 cases with HCC and the corresponding paracancerous tissues. The correlation between PTEN and hTERT was analyzed. The PTEN mRNA and protein expression was significantly lower in HCC, as compared with the paracancerous tissues (P<0.01), while those of hTERT demonstrated the opposite expression pattern (P<0.01). Additionally, both the PTEN and hTERT protein and mRNA levels demonstrated a significantly negative correlation with one another (P<0.05). In conclusion, PTEN and hTERT have different roles in the development of HCC. A significantly negative correlation between PTEN and hTERT gene expression indicates that hTERT activation and upregulation may be conferred by the loss of PTEN gene expression in HCC. The combined detection of PTEN and hTERT may provide critical clinical evidence for the diagnosis and biological behavior of HCC.

Non-Shelterin Telomere Genes Deregulation in Plasma Cell Disorders

Telomeres are essential structures that protect the ends of linear chromosomes. When telomeres become dysfunctional, critically short ends are sensed by the DNA repair machinery triggering senescence or apoptosis as well as creating interchromosomal fusions. Telomere structure and functions depend on the telomerase enzyme (hTERT, hTERC and DKC1) for elongation, on the sheterin complex that regulates telomere length (TL) and protects them against degradation and fusion, and on the non-shelterin complex. The latter is constituted by a set of multifunctional factors, including RPA1 and the MRE11-RAD50-NBS1 (MRN) complex, that facilitate telomerase-based telomere elongation. Particularly, RPA1 might be a crucial link between telomere homeostasis and the replication of chromosome ends as it regulate the telomerase action during the cell cycle. The role of MRN complex in telomere maintenance parallels its function in DNA repair. In telomerase-positive cells, a reduction of the MRN complex results in G-overhang shortening, suggesting MRN complex involvement in the recruitment or action of telomerase. Previous data from our group have provided the first evidence of modifications in the mRNA expression of shelterin members in plasma cell disorders (Panero et al, Mol Med 2010; 16: 471-8; Blood Cells Mol Dis 2014; 52: 134-9). The aim of the present study was to examine the expression profile of the non-shelterin complex: RPA1, RAD50, MRE11, NBS1 and DKC1 in patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Results were correlated with hTERT (telomerase catalytic subunit) mRNA levels, TL, and clinico-pathological characteristics of patients. Bone marrow samples from 70 cases: 35 MM (21 females; mean age: 67.9 years; range: 30-87 years; 35.3% stage III) and 35 MGUS (21 females; mean age: 68.7 years; range: 39-88 years) were studied. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Gene expression was quantified by Real-time Quantitative PCR, using Taq-Man methodology, and TL measurements were performed by Terminal Restriction Fragments. A significant increase in the mean mRNA levels of all non-shelterin genes in patients with MM respect to cases with MGUS was observed (Table 1). In order to evaluate whether the expression of these factors was associated to telomerase activity, hTERT transcripts were also quantified. An upregulation of telomerase expression, with significant differences between entities was found (p=0.03). In MM, higher DKC1 mRNA levels (0.10±0.02) in patients who over-expressed telomerase compared to patients with low hTERT expression (0.04±0.004) (p=0.008) was observed. No association between gene expression and TL or clinical parameters was found. To the best of our knowledge, our findings show for the first time a global modification in the expression of non-shelterin genes in MM and MGUS, suggesting that their upregulation in MM may occur as a result of the increasing need for DNA repair and telomerase recruitment, contributing to the progression of the disease. In addition, DKC1 was related to hTERTlevels in MM patients, supporting the role that DKC1 plays in telomerase complex stabilization and telomere maintenance.Table 1Expression profile of non-shelterin genes in patients with plasma cell disordersGenesMGUS (X±ES)MM (X±ES)p valueDKC10.036±0.0030.052±0.0050.02RAD500.060±0.0070.237±0.0640.0001MRE110.277±0.0400.545±0.1140.003NBS10.448±0.0660.894±0.1480.02RPA10.391±0.0440.591±0.0820.02 DisclosuresNo relevant conflicts of interest to declare.

Detection of telomerase reverse transcriptase mRNA in peripheral blood mononuclear cells of patients with liver failure.

Background:Telomerase activity is closely associated with the expression of human telomerase reverse transcriptase (hTERT) mRNA; although it can be induced in hepatocytes during liver regeneration, its dynamic change in patients with liver failure has remained unclear.Objectives:We investigated the variation and significance of hTERT mRNA expression in peripheral blood mononuclear cells (PBMCs) of the patients with liver failure.Patients and Methods:In this clinical experimental study, 76 Chinese patients were enrolled in the study between 2010 and 2012. The level of PBMCs hTERT mRNA was detected by relative quantitative real-time polymerase chain reaction (RT-PCR) in the samples taken before treatment and at seven-day intervals during a 28-day treatment period. The patients were divided into survivor and non-survivor groups according to the 3-months mortality after treatment. The dynamic variation of PBMCs hTERT mRNA was analyzed and its association with prognosis was assessed by the area under the receiver-operating characteristic curve.Results:The median level of PBMCs hTERT mRNA in survivors increased with treatment time and was significantly higher than the corresponding level in non-survivors after 14 days of treatment (P < 0.001). Subgroup analyses showed that the levels of PBMCs hTERT mRNA were remarkably higher in patients with acute-on-chronic liver failure than in those with chronic liver failure (P < 0.05). In patients with the same clinical type of liver failure, the level was markedly higher in survivors than in non-survivors after 14 days of treatment (P < 0.05); however, the levels were not significantly different between subgroups with different clinical type but the same prognosis. The sensitivity and specificity of PBMCs hTERT mRNA was high in evaluating the prognosis at day 14 and became much higher at days 21 and 28 post treatment. The expression of PBMCs hTERT mRNA had high sensitivity and specificity in evaluating the prognosis as early as day 14 post treatment and was significantly superior to the prognostic value of serum alpha-fetoprotein.Conclusions:The expression of PBMCs hTERT mRNA is closely associated with patient outcome, which indicates that hTERT mRNA in PBMCs might be useful as a prognostic biomarker of liver failure.

HCT116 colonospheres shows elevated expression of hTERT and β-catenin protein - a short report.

Clonospheres formed due to modified culture conditions are often studied for their stem cell like behaviour. The main objective of the current study is to compare the stem cell markers and link it to hTERT levels by monitoring their quantitative gene expression as they are potential targets for new generation combination therapeutics. In the present study we created stable colonospheres of Human colon cancer cell line HCT-116 long term culture conditions of Serum deprivation. Clonospheres formed after 15 days were collected by gentle and enzymatic dissociation was performed. Single cell suspension was obtained by mechanically dissociating the cells through a 22G needle. Single cells were replanted at a density 1200 cells/ml in Serum Free Medium in the 6 well plates for further passage. Passaging of cells was done at an interval of 8 days. The spheres formed were cyto-spun in special slides for Immunocytochemistry (ICC) studies for β-catenin protein and hTERT. The colonospheres were also processed for real time PCR expression studies for the same genes to confirm. In this present study, immunofluorescence studies revealed high β-catenin expression in the nucleus in colonospheres as compared to that of differentiated cancer cell line HCT-116 where the signal was localized mostly in the membranous and non-nuclear regions. Also increased TRF2 signal in colonospheres indicated higher activity of hTERT gene as TRF2 is the direct activator of hTERT to protect the telomere. Quantitative PCR studies showed that there was a significant over expression (p<0.05) at the mRNA level of the hTERT, TRF2, Rap1 genes along with the β-catenin over expression. Immunofluorescence analysis also revealed higher expression of CSC marker CD44 and ALDH1in colonospheres compared to the parental population. Clonospheres sub-population is showing higher degree of hTERT gene expression along with β-catenin when compared to the parental HCT-116 cancer cells. We also checked the co expression of other telomere maintenance genes mainly TRF 2 and Rap1 which also showed similar results. Therefore, we conclude that not only hTERT but possibly other Sheltrin proteins are regulated by β-catenin which is co expressed.

Differential telomerase expression in idiopathic pulmonary fibrosis and non-small cell lung cancer

Telomerase is a reverse transcriptase ribonucleo-protein (h-TERT) that synthesizes telomeric repeats using its RNA component (h-TERC) as a template. Telomerase dysfunction has been associated with both fibrogenesis and carcinogenesis. In this study, we aimed to evaluate the telomerase mRNA expression levels of both subunits (h-TERT and h-TERC) in lung tissue and bronchoalveolar lavage fluid (BALF) from patients with idiopathic pulmonary fibrosis (IPF) and non-small cell lung cancer (NSCLC), since there are indications of common pathogenetic pathways in these diseases. We prospectively examined lung tissue samples from 29 patients with IPF, 10 patients with NSCLC and 21 controls. Furthermore, we examined BALF samples from 31 patients with NSCLC, 23 patients with IPF and 12 control subjects. The mRNA expression for both h-TERT and h-TERC was measured by real-time RT-PCR. In the lung tissue samples, both h-TERT and h-TERC mRNA expression levels varied among the 3 groups (p=0.036 and p=0.002, respectively). h-TERT mRNA levels in the patients with IPF were lower compared with those in the controls (p=0.009) and patients with NSCLC (p=0.004). h-TERC mRNA levels in the patients with IPF were lower compared with those in the controls (p=0.0005) and patients with NSCLC (p=0.0004). In the BALF samples, h-TERT mRNA expression levels varied among the groups (p=0.012). More specifically, h-TERT mRNA levels in the patients with IPF were higher compared with those in the controls (p=0.03) and patients with NSCLC (p=0.007). The attenuation of telomerase gene expression in IPF in comparison to lung cancer suggests a differential role of this regulatory gene in fibrogenesis and carcinogenesis. Further functional studies are required in order to further elucidate the role of telomerase in these devastating diseases.

Hedgehog Signaling Regulates Telomerase Reverse Transcriptase in Human Cancer Cells

The Hedgehog (HH) signaling pathway is critical for normal embryonic development, tissue patterning and cell differentiation. Aberrant HH signaling is involved in multiple human cancers. HH signaling involves a multi-protein cascade activating the GLI proteins that transcriptionally regulate HH target genes. We have previously reported that HH signaling is essential for human colon cancer cell survival and inhibition of this signal induces DNA damage and extensive cell death. Here we report that the HH/GLI axis regulates human telomerase reverse transcriptase (hTERT), which determines the replication potential of cancer cells. Suppression of GLI1/GLI2 functions by a C-terminus truncated GLI3 repressor mutant (GLI3R), or by GANT61, a pharmacological inhibitor of GLI1/GLI2, reduced hTERT protein expression in human colon cancer, prostate cancer and Glioblastoma multiforme (GBM) cell lines. Expression of an N-terminus deleted constitutively active mutant of GLI2 (GLI2ΔN) increased hTERT mRNA and protein expression and hTERT promoter driven luciferase activity in human colon cancer cells while GANT61 inhibited hTERT mRNA expression and hTERT promoter driven luciferase activity. Chromatin immunoprecipitation with GLI1 or GLI2 antibodies precipitated fragments of the hTERT promoter in human colon cancer cells, which was reduced upon exposure to GANT61. In contrast, expression of GLI1 or GLI2ΔN in non-malignant 293T cells failed to alter the levels of hTERT mRNA and protein, or hTERT promoter driven luciferase activity. Further, expression of GLI2ΔN increased the telomerase enzyme activity, which was reduced by GANT61 administration in human colon cancer, prostate cancer, and GBM cells. These results identify hTERT as a direct target of the HH signaling pathway, and reveal a previously unknown role of the HH/GLI axis in regulating the replication potential of cancer cells. These findings are of significance in understanding the important regulatory mechanisms that determine the functions of HH/GLI signaling in cancer cells.

Abstract 741: CpG methylation abrogates KLF2-mediated transcriptional repression of telomerase in human T cells.

Abstract Many genes in tumor cells are DNA-methylated and transcriptionally repressed. Constitutive expression of the limiting catalytic subunit of telomerase, hTERT, is one of the common signatures of tumor cells, even though its promoter is DNA-methylated. On the other hand, in normal somatic cells the promoter is unmethylated and transcription of hTERT is strictly regulated. In this study, we examined transcriptional regulation of the hTERT gene in normal and tumor cells. We identified a novel promoter element, which bound the zinc-finger transcription factor Krüpple-like factor 2 (KLF2) in normal human T Lymphocytes. The binding of KLF2 to the hTERT promoter element paralleled repression of hTERT transcription in resting normal T cells. T cell growth induced KLF2 release from the element and hTERT transcription. Forced reduction of KLF2 expression by siRNA in normal resting T cells increased the level of endogenous hTERT mRNA. The hTERT promoter is methylated in the KLF2-positive human T leukemic cell line, Kit 225, and KLF2-negative human T leukemic cell line, Jurkat. Treatment with the DNA methyltransferase inhibitor, Zebularine, reduced levels of CpG methylation in the hTERT promoter, resulting in downregulation of hTERT mRNA expression in KLF2-positive Kit 225 cells, while the Zebularine treatment did not cause any appreciable effects on hTERT expression in KLF2-negative Jurkat cells. Methylation of CpG sequences in endogenous and exogenous hTERT promoter elements inhibited the KLF2 binding to the element. Furthermore, KLF2 binding to the promoter is associated with histone H3 repressive marks, indicating KLF2-mediated epigenetic silencing of the hTERT promoter. Our results suggest that KLF2 partly controls strict transcriptional regulation of the hTERT gene through association and dissociation with the promoter in normal T cells, but in tumor cells these important interactions are prevented by DNA methylation. Citation Format: Toshifumi Hara, Manami Yoshita, Masataka Nakamura. CpG methylation abrogates KLF2-mediated transcriptional repression of telomerase in human T cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 741. doi:10.1158/1538-7445.AM2013-741

Abstract 1790A: Hedgehog signaling at the level of GLI transcriptionally regulates hTERT in human cancer cells.

Abstract The HH signaling pathway is critical for normal embryonic development, tissue patterning and cell differentiation. Aberrant HH signaling is involved in multiple human cancers. HH signaling involves a multi-protein cascade activating the GLI proteins that transcriptionally regulate HH target genes. We have recently reported that HH signaling is essential for human colon cancer cell survival, and that inhibition of this signal induces DNA damage and extensive cell death. The current study demonstrates that HH signaling regulates human telomerase reverse transcriptase (hTERT), which determines the limitless replication potential of cancer cells. Suppression of both GLI1 and GLI2 functions by exogenous expression of a C-terminus truncated GLI3 repressor mutant (GLI3R), or by GANT61, a pharmacologic inhibitor of GLI1 and GLI2 transcriptional activity, reduced hTERT protein expression in human colon, prostate and brain cancer (glioblastoma, GBM) cell lines. Exogenous expression of a constitutively active mutant of GLI2, GLI2m, significantly increased hTERT transcription, protein expression, and hTERT promoter-luciferase activity, in human colon cancer cells. Exposure to GANT61 inhibited hTERT mRNA expression in human colon cancer cells. Insilico analysis of the hTERT promoter revealed 7 putative GLI binding sites suggesting a direct transcriptional mode of regulation of hTERT by GLI. Chromatin immunoprecipitation (ChIP) analysis with GLI1 or GLI2 antibodies precipitated fragments of the hTERT promoter in human colon cancer cells, indicating a direct interaction between GLI proteins and the hTERT promoter. The binding between GLI2 and the promoter of hTERT was significantly reduced upon exposure to GANT61. Of interest, exogenous expression of GLI1 or GLI2m in non-cancerous 293T cells failed to alter the levels of hTERT mRNA and protein, or hTERT promoter-luciferase activity. Further, ChIP analysis of GFP-tagged GLI2 did not precipitate the hTERT promoter in 293T cells, in contrast to events in malignant cells. GLI2m also increased telomerase activity in human colon cancer cells, while GANT61 reduced the telomerase activity in human colon, prostate and GBM cells. These results demonstrate that the HH signaling pathway directly regulates hTERT by direct interaction with GLI in cancer cells in contrast to non-transformed cells, and identify a previously unknown role of the HH/GLI axis in regulating the replication potential of cancer cells. These findings are of significance in understanding important regulatory mechanisms that determine the role of HH/GLI signaling in cancer cell survival. Citation Format: Tapati Mazumdar, Ranjodh Sandhu, Maha Qadan, Victoria Magloire, Akwasi Agyeman, Bibo Li, Janet A. Houghton. Hedgehog signaling at the level of GLI transcriptionally regulates hTERT in human cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1790A. doi:10.1158/1538-7445.AM2013-1790A

Combination of four gene markers to detect circulating tumor cells in the peripheral blood of patients with advanced lung adenocarcinoma using real-time PCR

The aim of this study was to establish a robust and reliable assay for the detection of circulating tumor cells (CTCs) in the peripheral blood (PB) of patients with advanced lung adenocarcinoma. We used real-time reverse transcription PCR (RT-PCR) to detect survivin, human telomerase reverse transcriptase (hTERT), cytokeratin-7 (CK-7) and thyroid transcription factor 1 (TTF-1) mRNA expression levels in 68 advanced lung adenocarcinoma patients and 30 healthy patients. Statistical analyses were additionally performed to examine the correlation between the mRNA expression levels of these markers with the clinicopathological features of advanced lung adenocarcinoma patients. The sensitivity of these four mRNA markers in the PB of advanced lung adenocarcinoma patients was 41.18, 61.76, 41.18 and 35.29%, respectively. The sensitivity of these four markers combined was 82.35%, which was significantly higher compared with single marker detection. Statistical analysis demonstrated that high expression levels of survivin, hTERT and TTF-1 mRNA are positively correlated with lymph node classification, and high expression levels of survivin, hTERT, CK7 and TTF-1 mRNA are positively correlated with distant metastasis (P<0.05). In addition, overexpression of these four mRNA markers is positively correlated with disease progression (P<0.05). Our data suggest that the combination of survivin, hTERT, CK-7 and TTF-1 mRNA markers may provide a valuable tool for CTC detection and is associated with disease progression in advanced lung adenocarcinoma patients.

DNA methy ltransferase 1 involves the up-regulation of human telomerase reverse transcriptase in RBE cell lines

Objective To explore the expression of human telomerase reverse transcriptase (hTERT) in intrahepatic cholangiocarcinoma cell line RBE transfected with hepatitis C virus core protein (HCVc) plasmid,and involvement of DNA methy ltransferase 1 (DNMT1) in the up-regulation of hTERT induced by HCVc.Methods Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to analyze the expression of hTERT and DNMT1 after the transfection of HCVc plasmid into RBE cells.5-Aza-2'-deoxycytidin (AZA) was used to inhibit DNMT1.Results hTERT expression level was higher in RBE cells after HCVc transfection and DNMT1 expression was up-regulated simultaneously.The mRNA and protein levels of hTERT were up-regulated by 3.457 ± 0.081 and 1.684 ± 0.020 respectively (P ＜ 0.05).And the expression levels of DNMT1 were up-regulated by 1.826 ± 0.060 and 2.513 ± 0.119 respectively (P＜ 0.05).After treatment with AZA,the mRNA and protein levels of hTERT were down-regulated by 2.755 ± 0.098 and 1.491 ± 0.045 respectively (P ＜ 0.05).Conclusion DNMT1 may take part in the up-regulation of hTERT induced by HCVc. Key words: Cholangiocarcinoma; Hepatitis C virus core protein; DNA methyltransferase; Telomerase

Effect of targeted silencing of hTERT mRNA by lentivirus-mediated siRNA on A549 lung cancer cells in vitro

In our present study, we took advantage of the characteristics of RNA interference technology, which can efficiently, stably, and specifically silence target genes, and designed a small interfering RNA (siRNA) that could specifically target hTERT mRNA. We used a lentiviral vector (LV) to deliver the hTERT siRNA into telomerase-positive A549 lung cancer cells and investigated the effect of hTERT siRNA on the hTERT mRNA levels, hTERT protein levels, cell proliferation, and apoptosis in the lung cancer cells. The results from quantitative PCR, Western blotting, and the MTT assay showed that the expression levels of both hTERT mRNA and protein in the cells were significantly decreased and that the cell proliferation rate started to significantly slow down at 48 h after transfection with hTERT-LV. Our study demonstrated that siRNA sequences specifically targeting hTERT mRNA, which were packaged into lentivirus particles and then used to transfect the lung cancer cell line A549, can specifically silence the mRNA of the target gene, hTERT, and then reduce the hTERT protein expression level, which, in turn, reduces cell proliferation, inhibits cell growth, and induces apoptosis.

Cell-Free Circulating Plasma hTERT mRNA Is a Useful Marker for Prostate Cancer Diagnosis and Is Associated with Poor Prognosis Tumor Characteristics

BackgroundSerum prostate-specific antigen (PSA) is the most widely used marker for diagnosing prostate cancer (PCa). It lacks specificity and predictive value, resulting in inaccurate diagnoses and overtreatment of the disease. The aim of this study was to assess the usefulness of plasma telomerase reverse transcriptase (hTERT) mRNA as a diagnostic and prognostic tool for PCa and its association with clinicopathological parameters of tumors.Principal FindingsPlasma hTERT mRNA levels were determined by qRT-PCR in 105 consecutive patients with elevated PSA levels and in 68 healthy volunteers. The diagnostic accuracy, the efficacy as a prognostic factor of biochemical recurrence and the association with tumor clinicopathological parameters of plasma hTERT mRNA and serum PSA tests were determined using univariate and multivariate analyses. The results show that plasma hTERT mRNA is a non-invasive biomarker for PCa diagnosis that shows higher sensitivity (85% vs. 83%), specificity (90% vs. 47%), positive predictive value (83% vs. 56%), and negative predictive value (92% vs. 77%) than serum PSA. Plasma hTERT mRNA is significantly associated with poor prognosis tumor clinicopathological parameters and is a significant independent predictor of PCa (p<0.0001). Univariate analysis identified plasma hTERT mRNA (but not serum PSA) as a significant prognostic factor of biochemical recurrence. Plasma hTERT mRNA Kaplan-Meier curves confirmed the significant differences between groups and patients with higher levels than the cut-off value showed diminished recurrence-free survival (p = 0.004), whereas no differences were observed with serum PSA (p = 0.38). Multivariate analysis indicated that plasma hTERT mRNA (but not serum PSA) and stage were significantly associated with biochemical recurrence.ConclusionsOverall, these findings indicate that hTERT mRNA is a useful non-invasive tumor marker for the molecular diagnosis of PCa, affording a greater diagnostic and prognostic accuracy than the PSA assay and may be of relevance in the follow-up of the disease.

The isoprenoid perillyl alcohol inhibits telomerase activity in prostate cancer cells

Isoprenoids are recognized for their ability to suppress carcinogenic processes in vivo and in vitro. We previously established that the isoprenoid, perillyl alcohol, acted mechanistically on translation of specific proteins through modulation of mechanistic target of rapamycin (mTOR) signaling. Telomerase-the enzyme responsible for immortalizing cells through the addition of telomeric repeats-is de-repressed early in an aspiring cancer cell. Here the effects of biologically-relevant concentrations and short incubations (1-16 h) of perillyl alcohol or the mTOR inhibitor, rapamycin, on telomerase activity were examined in prostate cancer cell lines. A rapid suppression of telomerase activity was observed (from ∼65% to >95%) determined by real-time quantitative telomerase repeat amplification protocol and confirmed by polyacrylamide gel-analysis. Using real-time reverse transcriptase-PCR, we demonstrated that human telomerase reverse transcriptase (hTERT) mRNA levels were unaltered. Western blot analysis revealed that hTERT protein levels decreased in response to perillyl alcohol or rapamycin. This decrease was partially blocked by pretreatment with a proteasome inhibitor MG-132, indicating that proteasomal degradation contributed to the loss of hTERT protein. No change in hTERT phosphorylation at Ser824 was observed, indicating the absence of cellular hTERT protein redistribution. These findings provide evidence for a unique link between nutrient- and macrolide-mediated regulation of mTOR and hTERT, a key enzyme that regulates DNA structure and stability.

Preoperative peripheral blood human telomerase reverse transcriptase mRNA concentration is not a prognostic factor for resection of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) often recurs after complete surgical resection. Detection of markers of residual circulating cancer cells may predict postoperative HCC recurrence. Human telomerase reverse transcriptase (hTERT) mRNA may be a candidate tumor marker. We prospectively assessed the expression patterns and prognostic value of preoperative peripheral blood hTERT mRNA in patients with HCC undergoing hepatic resection (n=17) or liver transplantation (n=6). As controls, we assessed hTERT mRNA in patients with liver cirrhosis without HCC (n=6) and in living liver donors (n=4). Concentrations of hTERT mRNA were measured by real-time quantitative reverse transcription polymerase chain reaction (RTPCR). No significant difference was observed in the levels of hTERT mRNA between the HCC and control groups. Only alpha-fetoprotein ≥400ng/mL was associated with greater expression levels of hTERT mRNA. At a median follow-up of 30 months, HCC recurred in 10 of 17 resected patients, but in none of the 6 liver transplant recipients. hTERT mRNA concentration was not associated with HCC recurrence after either resection or liver transplantation. Peripheral blood hTERT mRNA concentration is not a likely marker for the diagnosis or prognosis of HCC, especially in patients undergoing resection. Owing to the small number of transplanted patients assessed, the clinical significance of hTERT mRNA concentration was not objectively verified, suggesting the need for a study in larger numbers of HCC patients undergoing liver transplantation.

Multiple factors influencing the release of hTERT mRNA from pancreatic cancer cell lines inin vitroculture

Since telomerase expression is highly prevalent in human cancers, the quantitation of serum/plasma hTERT (human telomerase reverse transcriptase) mRNA levels may be useful for early detection of PCa (pancreatic cancer). To analyse the correspondence between exhTERT (extracellular hTERT) mRNA levels and hTERT expression, we designed a cell culture system to investigate factors modulating the extracellular levels of hTERT mRNA in media conditioned by eight PCa cell lines. We found that the level of exhTERT mRNA was dependent on cell growth rate. MIAPaCa-2, PANC-1, KLM-1 and PK-9 cells expressed high levels of exhTERT mRNA, independent of cell density, whereas proliferating PK-59, BxPC-3 and PK-45H cells released low levels of exhTERT mRNA. The augmented release of mRNA by spontaneous dead MIAPaCa-2 cells was further increased at postconfluence. In Capan-1 cells, low correspondence of marker was also due to RNase secretion. Upon reaching confluence, some PCa cell lines showed down-regulation of hTERT expression. Following cell-cell adhesion, as shown by E-cadherin engagement, PK-59 cells showed levels of extracellular message below the limits of detection, a loss not due to an increase in message degradation. These results suggest that the levels of exhTERT mRNA in the medium of PCa cell lines are altered not only in response to cell growth rate and cell destruction, but are responsive to extracellular cues such as RNases and cell density. A cell-free assay for exhTERT mRNA may therefore not be useful for early detection of PCa.

Plasma hTERT mRNA discriminates between clinically localized and locally advanced disease and is a predictor of recurrence in prostate cancer patients

Introduction: Since the introduction of prostate-specific antigen (PSA) testing, new prostate cancer (PCa) patients are diagnosed earlier and most have localized and locally advanced disease. Current diagnosis methods lack specificity and sensitivity, leading to overdiagnosis and overtreatment of patients with low-risk organ-confined localized disease. Therefore, new non-invasive molecular tools are needed to discriminate between localized and locally advanced disease.Methods: Plasma telomerase reverse transcriptase (hTERT) mRNA levels were determined by qRT-PCR in 49 patients with localized and locally advanced PCa. Diagnostic accuracy and efficacy as a prognostic factor of biochemical recurrence of plasma hTERT mRNA were determined using univariate and multivariate analyses and compared with conventional tumor markers.Results: Patients with locally advanced disease had significantly (p < 0.05) higher plasma hTERT mRNA and serum PSA levels than those with localized disease. Plasma hTERT mRNA test showed lower sensitivity (83% vs. 100%), higher specificity (73% vs. 43%), AUC ROC curve (0.911 vs. 0.757), and positive likelihood ratios (6.17 vs. 1.76) than the PSA assay in discriminating between localized and locally advanced disease. At multivariate analysis, plasma hTERT mRNA levels and age but not PSA showed a positive trend (p = 0.05) in the risk of locally advanced PCa. On univariate analysis, plasma hTERT mRNA and serum PSA were identified as significant prognostic factors of biochemical recurrence. Using ROC curves and the appropriate cutoff, both tests showed high sensitivity (85%) and specificity (72%). Kaplan–Meier curves confirmed the significant differences between the groups and patients with higher levels than the cutoff value showed diminished recurrence-free survival (p < 0.05). At multivariate analysis, Gleason score and PSA were the strongest factors associated with biochemical recurrence (p < 0.05), whereas hTERT mRNA did not reach statistical significance, although a positive trend was observed (p = 0.09).Conclusion: Plasma hTERT mRNA quantification can be both a useful non-invasive tumor marker for discriminating between localized and locally advanced PCa, as well as a prognostic factor of recurrence at the molecular level.

The isoprenoid perillyl alcohol inhibits telomerase activity in prostate cancer cells

Plant‐derived isoprenoids suppress carcinogenic processes in vivo and in vitro. Previously we established that the isoprenoid, perillyl alcohol (POH), acted mechanistically on translation of specific proteins through modulation of mammalian target of rapamycin (mTOR) signaling. Telomerase–the enzyme responsible for immortalizing cells through addition of telomeric repeats–is derepressed early in an aspiring cancer cell. We examined effects of biologically‐relevant concentrations and short incubations (1–16 h) of POH or the mTOR inhibitor, rapamycin, on telomerase activity in prostate cancer cell lines. A rapid suppression of telomerase activity (from 70 to &gt;95%) was observed by real‐time quantitative telomerase repeat amplication protocol and confirmed by polyacrylamide gel analysis. However, human telomerase reverse transcriptase (hTERT) mRNA levels were unaltered as determined by quantitative RT‐PCR. Western blot analysis revealed that hTERT protein levels decreased in response to POH or rapamycin, but E3 ligase Makorin‐1–which is thought to signal proteolytic breakdown of hTERT–was not detectable. No change in hTERT phosphorylation at Ser824 was observed, and no cellular redistribution of hTERT protein was found. These findings provide evidence for a unique link between nutrient‐ and macrolide‐mediated regulation of mTOR and hTERT‐a key factor regulating DNA structure and stability.

Complementary treatment of siTERT for improving the antitumor effect of TERT-specific I-131 therapy

Sodium iodide symporter (NIS)-based radionuclide therapy provides an effective means of treating malignant tumors. However, it is sometimes inadequate because of limited effects on radio-resistant tumors, and thus, combination therapies with other therapeutic options have been requested to enhance its efficacy. Human telomerase reverse transcriptase (hTERT) has been reported to be involved in the progression of most cancers and also been viewed as a good candidate for targeting tumor. Application of TERT-specific radionuclide therapies using NIS gene transfer have been reported to treat TERT-positive tumors, but this approach only demonstrated tumor regression rather than eradication. As inhibiting TERT expression by introducing the hTERT-specific shRNA (siTERT) has been suggested as a therapeutic option, we investigated the complementary role of siTERT treatment after the TERT-specific I-131 therapy and its possibility as a novel anticancer therapeutic strategy. Retroviruses containing TERT promoter/NIS for TERT specific Radionuclide therapy and siTERT for TERT targeting antisense therapy were produced. Hep3B cells expressing TERT specific NIS (Hep3B-TERT/NIS) were xenografted into nude mouse and visualized with micro-SPECT/CT for monitoring NIS activity. The levels of hTERT mRNA, protein and its activity were confirmed by RT-PCR, Western blotting and Telomerase repeat amplification protocol assay. Cell proliferation was monitored by MTT assay and induced apoptosis was confirmed by Annexin-V-PI staining. Therapeutic effects of I-131 and/or siTERT were evaluated by clonogenic assay and mouse tumor model. Reduction of hTERT mRNA, protein and TERT activity by siTERT were observed in Hep3B-TERT/NIS cells. The viabilities of the infected cells were significantly decreased to 50% versus siScramble treated controls. The early apoptotic cell population was increased by siTERT. The survival rates of cells treated with siTERT or I-131 alone were 72.4±7.6% and 56.2±5.2%, respectively. However, the survival rate of cells treated with I-131 and siTERT were decreased to 22.1±2.8%. From mouse xenograft model, we also found that the siTERT gene therapy showed synergism to the radioiodine therapy for reducing tumor growth in vivo. Our Results suggested that complementary siTERT gene therapy offers a novel strategy of cancer therapy to improve the therapeutic efficacy of TERT-specific I-131.

The AAA-ATPase NVL2 is a telomerase component essential for holoenzyme assembly

Continued cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity. Although the core enzyme includes a telomerase reverse transcriptase (TERT) and a telomerase RNA component (TERC), a number of auxiliary proteins have been identified to regulate telomerase assembly, localization, and enzymatic activity. Here we describe the characterization of the AAA-ATPase NVL2 as a novel hTERT-interacting protein. NVL2 interacts and co-localizes with hTERT in the nucleolus. NLV2 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT. Depletion of endogenous NVL2 by small interfering RNA led to a decrease in hTERT without affecting the steady-state levels of hTERT mRNA, thereby reducing telomerase activity, suggesting that NVL2 is an essential component of the telomerase holoenzyme. We also found that ATP-binding activity of NVL2 is required for hTERT binding as well as telomerase assembly. Our findings suggest that NVL2, in addition to its role in ribosome biosynthesis, is essential for telomerase biogenesis and provides an alternative approach for inhibiting telomerase activity in cancer.