Human Telomerase Reverse Transcriptase mRNA Research Articles

Abstract 2743: Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma

Abstract Background: The telomerase-specific oncolytic adenovirus Telomelysin along with the histone deacetylase inhibitor AR42 have both demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies has been reported to be a viable approach for treating various cancers. We investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Method: HCC cell lines (PLC5 and Hep3B) were tested and median effect analysis was performed to estimate potential synergistic antiproliferative effects. Apoptosis was assessed by flow cytometry and Western blotting. The expression of coxsackievirus and adenovirus receptor (CAR) was measured through flow cytometry and immunofluorescence staining. Human telomerase reverse transcriptase (hTERT) mRNA expression was assessed by reverse transcription polymerase chain reaction. Western blotting was conducted to examine the activation of pivotal signaling pathways and the interferon (IFN) response. HCC cells constitutively overexpressing Akt were cloned for molecular mechanism verification. MK-2206 was used to inhibit the activation of Akt signaling. In vivo efficacy was determined through xenograft models of human HCC. Results: Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the CAR and the mRNA levels of hTERT, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Conclusions: Combination therapy with Telomelysin and AR42 demonstrates synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted. Keywords: Hepatocellular carcinoma, oncolytic adenovirus, Telomelysin, HDAC inhibitor, Akt Citation Format: Zhong-Zhe Lin, Mickey C-T. Hu, Chiun Hsu, Yao-Ming Wu, Yen-Shen Lu, Ja-An Annie Ho, Shiou-Hwei Yeh, Pei-Jer Chen, Ann-Lii Cheng. Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2743.

Lab-on-chip assay of tumour markers and human papilloma virus for cervical cancer detection at the point-of-care

Cervical cancer affects over half a million people worldwide each year, the majority of whom are in resource-limited settings where cytology screening is not available. As persistent human papilloma virus (HPV) infections are a key causative factor, detection of HPV strains now complements cytology where screening services exist. This work demonstrates the efficacy of a handheld Lab-on-Chip (LoC) device, with an external sample extraction process, in detecting cervical cancer from biopsy samples. The device is based on Ion-Sensitive Field-Effect Transistor (ISFET) sensors used in combination with loop-mediated isothermal amplification (LAMP) assays, to amplify HPV DNA and human telomerase reverse transcriptase (hTERT) mRNA. These markers were selected because of their high levels of expression in cervical cancer cells, but low to nil expression in normal cervical tissue. The achieved analytical sensitivity for the molecular targets resolved down to a single copy per reaction for the mRNA markers, achieving a limit of detection of 102 for hTERT. In the tissue samples, HPV-16 DNA was present in 4/5 malignant and 2/5 benign tissues, with HPV-18 DNA being present in 1/5 malignant and 1/5 benign tissues. hTERT mRNA was detected in all malignant and no benign tissues, with the demonstrated pilot data to indicate the potential for using the LoC in cervical cancer screening in resource-limited settings on a large scale.

Berberine Inhibits Telomerase Activity and Induces Cell Cycle Arrest and Telomere Erosion in Colorectal Cancer Cell Line, HCT 116.

Colorectal cancer (CRC) is the most common cancer among males and females, which is associated with the increment of telomerase level and activity. Some plant-derived compounds are telomerase inhibitors that have the potential to decrease telomerase activity and/or level in various cancer cell lines. Unfortunately, a deeper understanding of the effects of telomerase inhibitor compound(s) on CRC cells is still lacking. Therefore, in this study, the aspects of telomerase inhibitors on a CRC cell line (HCT 116) were investigated. Screening on HCT 116 at 48 h showed that berberine (10.30 ± 0.89 µg/mL) is the most effective (lowest IC50 value) telomerase inhibitor compared to boldine (37.87 ± 3.12 µg/mL) and silymarin (>200 µg/mL). Further analyses exhibited that berberine treatment caused G0/G1 phase arrest at 48 h due to high cyclin D1 (CCND1) and low cyclin-dependent kinase 4 (CDK4) protein and mRNA levels, simultaneous downregulation of human telomerase reverse transcriptase (TERT) mRNA and human telomerase RNA component (TERC) levels, as well as a decrease in the TERT protein level and telomerase activity. The effect of berberine treatment on the cell cycle was time dependent as it resulted in a delayed cell cycle and doubling time by 2.18-fold. Telomerase activity and level was significantly decreased, and telomere erosion followed suit. In summary, our findings suggested that berberine could decrease telomerase activity and level of HCT 116, which in turn inhibits the proliferative ability of the cells.

GDF15 as a biomarker of ageing.

The ageing process is accompanied by the gradual development of chronic systemic inflammation (inflamm-ageing). Growth differentiation factor 15 (GDF15) is associated with inflammation and known to be a stress-induced factor. The present study aimed to explore the association of GDF15 with ageing. In this cross-sectional study, serum GDF15, hematological parameters, and biomedical parameters were determined in 120 healthy individuals (23-83years old, males). Three telomere related parameters, including telomere length, telomerase activity, and the expression of human telomerase reverse transcriptase (hTERT) mRNA were also quantified. Our results showed that the older group has a higher levels of GDF15 and lower expression of hTERT mRNA, and PBMC telomerase activity (p<0.001). In individuals with high GDF15 levels, they were older, and presented with the lower level of hTERT mRNA and T/S ratio (p<0.01). Spearman correlation analysis shows that GDF15 positively correlated with age (r=0.664, p<0.001), and negatively correlated with telomere length (r=-0.434, p<0.001), telomerase activity (r=-0.231, p=0.012), and hTERT mRNA (r=-0.206, p=0.024). Furthermore, in multivariate regression analysis, GDF15 levels showed a statistically significant linear and negative relationship with PBMC telomerase activity (β-coefficient=-0.583, 95% CI -1.044 to -0.122, p=0.014), telomere length (β-coefficient=-0.200, 95% CI -0.305 to -0.094, p<0.001), and hTERT mRNA (β-coefficient=-0.207, 95% CI -0.312 to -0.102, p<0.001) after adjusting for confounders. These results support that circulating GDF15 is the potential biomarker of ageing that may influence the risk and progression of multiple ageing conditions.

Detection of mRNA of Telomerase Protein in Benign Naevi and Melanomas Using RNAscope.

Telomerase is reactivated in most cancers and is possibly an early driver event in melanoma. Our aim was to test a novel in situ hybridization technique, RNAscope, for the detection of human telomerase reverse transcriptase (hTERT) mRNA in archival formalin-fixed, paraffin-embedded (FFPE) tissue and to compare the mRNA expression of melanomas and benign naevi. Furthermore, we wanted to see if hTERT mRNA could be a diagnostic or prognostic marker of melanoma. In situ hybridization for the detection of hTERT mRNA was performed on FFPE tissue of 17 melanomas and 13 benign naevi. We found a significant difference in the expression of hTERT mRNA between melanomas and benign naevi (P<0.001) and the expression of hTERT mRNA correlated with Breslow thickness (ρ=0.56, P=0.0205) and the Ki67 proliferation index (ρ=0.72, P=0.001). This study showed that RNAscope was a reliable in situ hybridization method for the detection of hTERT mRNA in FFPE tissue of melanomas and benign naevi. hTERT mRNA was more abundantly expressed in melanomas compared with benign naevi, but cannot be used solely as a diagnostic marker due to an overlap in expression. The hTERT mRNA expression in melanomas correlated with the prognostic markers Breslow thickness and the Ki67 index indicating a prognostic potential of hTERT mRNA.

Diagnostic value of detection of peripheral blood human telomerase reverse transcriptase mRNA and four tumor markers in patients with gastric cancer

Objective To evaluation the value of single and combined detection of peripheral blood human telomerase reverse transcriptase (hTERT) mRNA and tumor markers carcinoembryonic antigen (CEA), carbohydrate antigen 72-4 (CA72-4), CA19-9 and CA125 in the diagnosis of gastric cancer. Methods A total of 48 patients diagnosed with gastric cancer from June 2017 to October 2018 in Baotou Tumor Hospital were enrolled in the study group. Fifty healthy subjects were selected as healthy control group. The peripheral venous blood samples were collected from all subjects. After the synthesis of hTERT cDNA by reverse transcription, real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used for the amplification of hTERT gene fragment. The tumor markers CEA, CA72-4, CA19-9 and CA125 were quantitatively detected by electrochemiluminescence instrument. The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio of individual markers or joint detection were calculated. The sensitivity and specificity of joint testing items were compared. Results There were statistically significant differences in the positive rates of hTERT mRNA (P 0.05). The expression of hTERT mRNA was correlated with TMN stage, lymph node metastasis and distant metastasis (all P 0.05). The results of diagnostic indices showed that the hTERT mRNA had high sensitivity (84.8%), specificity (82.7%), accuracy (83.7%), positive predictive value (81.2%) and positive likelihood ratio (4.90). CA72-4 also had high specificity (61.2%), accuracy (64.3%), and positive predictive value (45.8%), which were second only to the hTERT mRNA. The diagnostic performance of CA125 combined with CEA, CA72-4, CA19-9 and peripheral blood hTERT mRNA was not significantly different from the latter four combined detection (P > 0.05). The sensitivity and specificity of combined detection of traditional tumor markers CEA, CA72-4 and CA19-9 were 62.5% and 80.0%. The sensitivity and specificity of combined detection of hTERT mRNA, CEA, CA72-4 and CA19-9 could be change to 90.0% and 67.5%. The difference in sensitivity between the two combined detection groups was statistically significant (P = 0.019), and the difference in specificity was not statistically significant (P > 0.05). Conclusion The comprehensive evaluation index of peripheral blood hTERT mRNA is better than the traditional gastric tumor markers, and it is expected to become a new marker for the diagnosis of gastric cancer. Key words: Stomach neoplasms; Diagnosis; Telomerase reverse transcriptase; Tumor markers, biological; Polymerase chain reaction

Template-ready PCR method for detection of human telomerase reverse transcriptase mRNA in sputum

Human telomerase reverse transcriptase (hTERT) mRNA in tissue is a biomarker of lung cancer, but hTERT mRNA in sputum had not been successfully detected with conventional reverse transcription PCR methods. Here, we developed a novel PCR protocol: Template-Ready PCR (TRPCR), to detect sputum hTERT mRNA, in which probes serve as templates of amplification. While free probes and dsDNA were removed in template preparation through aspiration and restriction digestion, probes that formed into heterocomplex with target RNA remained intact for PCR amplification. By fishing out the heterocomplex and amplifying the probes, TRPCR achieved sensitivity higher than reverse transcription-quantitative PCR (RT-qPCR). ROC curve of sputum hTERT mRNA by TRPCR assay showed the discrimination in high sensitivity and specificity between patients with lung cancer and lung cancer-free donors at the PCR Ct cutoff of 33. We further validated this approach through TRPCR assay of sputum from 858 lung cancer patients and 480 non-malignant pulmonary disease patients. 722 (84.2%) cases from 858 with lung cancer patients were detected as positive, whereas 461 (96.0%) cases from 480 non-malignant pulmonary disease patients were detected as negative, suggesting that TRPCR assay of sputum hTERT mRNA can serve as a non-invasive molecular diagnosis of lung cancer.

Nörofibromatozis tip1 hastalarında telomeraz aktivitesi

Purpose: In various studies low telomerase activity have been shown to have a protective role in some types of cancer. Patients with neurofibromatosis type-1 (NF1) are at risk of developing certain types of cancer. Prognosis is generally better in patients with NF1 than those without NF1. In the present study, we aimed to measure telomerase activity in patients with NF1 and in controls.Materials and Methods: Telomerase activity was investigated in peripheral blood samples with human telomerase reverse transcriptase (hTERT) mRNA by using real time reverse transcriptase polymerase chain reaction (RT-PCR) method and Light Cycler 480 system.Results: hTERT expression was investigated in blood samples of 48 patients with a diagnosis of NF1 and in 37 controls. Telomerase activity was positive in 36 of 48 (75%) patients with NF1 and 23 of 37 patients (73%) without NF1. Telomerase activity was positive in 31 of 36 patients (86%) with NF1 having a benign or malignant tumor, whereas it was positive in 5 of 12 NF1 patients (41.6%) without a tumor. Conclusion: Detection of hTERT expression in patients with NF1 can be used as a useful marker for tumorigenesis. Additional studies need to be done to know whether detection of hTERT expression in low-grade tumors would help predict progression to high-grade tumors.

Abstract P267: Aging &amp; Endothelial Cell Senescence: Does Prolylcarboxypeptidase Predict Endothelial Dysfunction?

Changes due to cellular senescence may lead to the formation of a unique cellular morphology such as the transformation of endothelium from an anticoagulant to a potentially prothrombotic surface. Investigations were performed to determine whether the ratio of prolylcarboxypeptidase (PRCP) gene expression to the rate of telomere shortening in vascular endothelial cells would predict endothelial dysfunction and subsequent endothelial surface alteration from anticoagulant to potentially prothrombotic surface. Here, we present evidence for a novel mechanism of senescence-induced cell-surface expression of PRCP involving activation of the plasma kallikrein-kinin system (KKS). We found that the abilities of human pulmonary vein endothelial cells (HPVECs) to express PRCP first incrementally increased at early passages and then decreased after passage 18, in contrast to changes in average human telomerase reverse transcriptase (hTERT) mRNA expression levels. In agreement with the changes in average PRCP expression, incubation of prekallikrein (PK) and high molecular weight kininogen (HK) plasma proteins resulted in increased cleavage of HK, higher enzymatic activities of kallikrein, and increased liberation of bradykinin (BK). Furthermore, the alterations in PK and BK generation was prevented using PRCP-siRNA or PRCP inhibitor (z-Pro- prolinal) that blocked PK activation in passage 18 by 60% and 80% respectively. SDD31 that interferes with the binding of PK on HK reduced kallikrein generation by 90%. There was a direct correlation between PRCP-dependent BK generation and cell permeability. Our results were supported when we found that nitric oxide (NO), a downstream product of the PRCP-catalyzed HK-PK system, also shared the same peak in formation as PRCP. In contrast, increased eNOS expression remained unchanged after passage 12. While β-Gal rose after passage 18, calnexin expression like PRCP dropped significantly in passage 30. These data are the first to demonstrate the inverse relationship between hTERT and PRCP expression prior to an apparent onset of EC replicative senescence, which could have profound implications as a prognostic tool for predicting disease progression and cardiovascular event rates.

Role of telomerase expression in T cell differentiation and proliferation

Abstract To understand the role of telomerase in T cell differentiation and function, we analyzed human telomerase reverse transcriptase (hTERT) mRNA expression and telomerase activity in six T cell subsets from 85 healthy human donors (aged 17–82 years old). We found that levels of hTERT mRNA and telomerase activity were higher in CD4 T cell subsets as compared to corresponding CD8 T cell subsets, and decreased from naïve (TN) to memory (central, TCM and effector, TEM). In all six subsets, in vitro activation with anti-CD3/CD28 antibody-coated beads led to increased amounts of hTERT mRNA (from 12 to 105-fold) as well as telomerase enzymatic activity, while the differences in hTERT amounts and telomerase activity among subsets remained in the same order observed prior to stimulation. We next analyzed the impact of different amounts of hTERT and induced telomerase activity on proliferation and lifespan of these subsets in vitro. We found that proliferation and survival were most robust in CD4 TN and poorest in CD8 TEM, correlating with their respective levels of hTERT mRNA and telomerase activity both pre-activation and over the course of a 15-day culture following a single stimulation. These findings suggest that greater hTERT expression and telomerase activity promote T cell viability and proliferative potential, and that differentiation is associated with loss of proliferative potential in T cells. We are currently analyzing whether inhibition of hTERT and telomerase activity will lead to reduced proliferation and viability in TN cells. This research was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging.

Expression of human telomerase reverse transcriptase mRNA in colorectal cancer patients and its clinical significance

Objective To investigate the expression of human telomerase reverse transcriptase (hTERT) mRNA in colorectal cancer patients, and to explore its clinical significance. Methods The clinical data of 80 patients with colorectal cancer who received treatment in our hospital from February 2014 to August 2015 were retrospectively analyzed. At the same time, the clinical data of 80 healthy adults were selected as control. To observe the expression of hTERT mRNA in colorectal tissue of two groups of patients, and to compare the expression of hTERT mRNA in colorectal tissue of colorectal cancer patients with different clinicopathological features, and to analyze the factors influencing the expression of hTERT mRNA in colorectal cancer patients. Results The positive expression rate of hTERT mRNA in colorectal tissue of colorectal cancer patients was 87.50%, which was significantly higher than that in normal control group (6.25%). Abdominal lymph node metastasis, histological type and tube adenocarcinoma and TNM stage Ⅲ+ Ⅳ, the degree of differentiation for poorly differentiated colorectal cancer colorectal tissue the positive expression rate of hTERT mRNA was higher (χ2=4.068, 4.898, 8.447, 6.154; P=0.044, 0.027, 0.004, 0.013), different age, gender, colorectal tissues of patients with colorectal cancer hTERT mRNA expression of tumor size and location was not significant difference (χ2=2.593, 0.258, 0.878, 0.152, 1.755; P=0.107, 0.612, 0.349, 0.696, 0.185). Abdominal lymph node metastasis and histological type were the risk factors of hTERT mRNA positive rate in patients with colorectal cancer. The odds ratio (OR) values were 4.115 and 3.985, respectively. Conclusion The expression of hTERT mRNA in colorectal tissue of patients with colorectal cancer was higher, and the existence of patients with lymph node metastasis, tumor stage and histological type are closely related, as an important index for clinical screening of colorectal cancer. Key words: Colorectal cancer; Human telomerase reverse transcriptase; Logistic regression analysis

Effect of Achillea wilhelmsii extract on expression of the human telomerase reverse transcriptase mRNA in the PC3 prostate cancer cell line

Effect of Achillea wilhelmsii extract on expression of the human telomerase reverse transcriptase mRNA in the PC3 prostate cancer cell line

Alternative splicing of telomerase catalytic subunit hTERT generated by apoptotic endonuclease EndoG induces human CD4+ T cell death

Telomerase activity is regulated by alternative splicing of its catalytic subunit human Telomerase Reverse Transcriptase (hTERT) mRNA. Induction of a non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of the apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. A strong correlation was identified between EndoG expression levels and hTERT splice variants in human CD4+ and CD8+ T lymphocytes. Overexpression of EndoG in CD4+ T cells down-regulated the expression of the active full-length hTERT variant and up-regulated expression of the non-active spliced variant. A reduction in full-length hTERT transcripts down-regulated telomerase activity. Long-term in vitro cultivation of EndoG-overexpressing CD4+ T cells led to dramatically shortened telomeres, conversion of cells into a replicative senescence state, and activation of the BCL2/BAX-associated apoptotic pathway finally leading to cell death. These data indicated the participation of EndoG in alternative mRNA splicing of the telomerase catalytic subunit hTERT, regulation of telomerase activity and determination of cell fate.

Effect of Achillea wilhelmsii extract on expression of the human telomerase reverse transcriptase mRNA in the PC3 prostate cancer cell line.

Evidence has indicated that human telomerase reverse transcriptase (hTERT) was overexpressed in prostate cancer (PCa). Achillea wilhelmsii (AW) is a plant that has been traditionally used for its medicinal properties. The aim of current study was to evaluate the effects of AW extract on a PCa cell line. The cytotoxic activity of the hydroalcoholic extract of AW was studied on the PCa PC3 cell line using MTT assay. Flow cytometry was used to evaluate the effects of the extract on the apoptosis. The expression of hTERT mRNA was analyzed by the reverse transcription-quantitative polymerase chain reaction method. The ELISA method was used to measure the levels of telomerase enzyme. The hydroalcoholic AW extract demonstrated the appropriate inhibitory effect in 150 µg/ml concentration (IC50) on PC3 cell line following 48 h treatment. Treatment of the PC3 cells with AW resulted in a significant increase in early and late apoptotic cells and a decrease in live cells (P<0.001), in a dose-dependent manner. Moreover, the early apoptotic cells were significantly higher than late apoptotic cells. The hTERT mRNA expression was decreased following 24 h treatment of AW extract, although it was not different between 2, 4, 8 and 12 h treatments or 24, 48 and 72 h treatments. In addition, the hTERT concentration was significantly decreased following 24 h treatment of AW extract with the marginal P-value. There was no significant difference regarding hTERT concentration between 2, 4, 8 and 12 h treatments or 24, 48 and 72 h treatments. The hydroalcoholic extract of AW induced potent antiproliferative and apoptotic effects in PC3 cell line, which could be explainable by its high potency to inhibit expression of the prominent oncogene hTERT in PCa. Therefore, targeting telomerase represents a promising strategy for PCa therapy, and AW may have considerable potential for development as a novel natural anticancer agent.

Characterisation of blood-derived exosomal hTERT mRNA secretion in cancer patients: a potential pan-cancer marker

Background:Telomerase (human telomerase reverse transcriptase (hTERT)) is considered a hallmark of cancer. The aim of our study was to evaluate the feasibility of the detection of hTERT transcripts in serum as a ‘pan-cancer’ diagnostic method.Methods:Human telomerase reverse transcriptase mRNA levels were determined in serum and serum-derived exosomes from 133 patients with different malignancies and 45 healthy controls. In four patients hTERT mRNA levels were measured in different clinical stages.Results:Human telomerase reverse transcriptase transcript was absent in all controls and was variably detected in 67.5% of patients with all cancer types. A correlation between hTERT transcript levels and the clinical course was found in several cases.Conclusions:Human telomerase reverse transcriptase mRNA levels may reflect the tumour burden and the clinical status of the patient. In patients with detectable levels, this assay may potentially serve as a diagnostic and follow-up ‘pan-cancer’ marker. Owing to the large variety of patients and small sample size in each diagnosis, the statistical power is limited and will be explored further in larger groups.

Brief Report: Differential Effects of Tenofovir, Abacavir, Emtricitabine, and Darunavir on Telomerase Activity In Vitro.

In vitro, tenofovir and abacavir induced a significant dose-dependent inhibition of telomerase activity at therapeutic concentrations in peripheral blood mononuclear cells of healthy subjects. Median inhibition of telomerase activity by tenofovir at 0.5 and 1 μM was 29% [Interquartile range (IQR) 29%-34%, P = 0.042] and 28% (IQR 28%-41%, P = 0.042), respectively. Abacavir inhibition was 12% (IQR 9%-13%, P = 0.043) at 3 μM and 14% (IQR 10%-29%, P = 0.043) at 10 μM. Tenofovir and abacavir did not change human telomerase reverse transcriptase (hTERT) levels or mRNA levels of other telomerase complex genes. Exposure to emtricitabine or darunavir did not affect telomerase activity, hTERT protein levels, or mRNA levels of telomerase/shelterin genes.

Assessment Effects of Resveratrol on Human Telomerase Reverse Transcriptase Messenger Ribonucleic Acid Transcript in Human Glioblastoma.

Background:Glioblastoma (GBM) is the most common and aggressive brain tumor, which has a poor prognosis despite the advent of different therapeutic strategies. There are numerous molecular biomarkers to contribute diagnosis, prognosis, and prediction of response to the current therapy in GBM. One of the most important markers that are potentially valuable is immortalization-specific or immortalization-associated marker named “hTERT messenger ribonucleic acid (mRNA)” the key subunit of telomerase enzyme, which is expressed in more than 85% of cancer cells, in spite of the majority of normal somatic cells. In this study, we investigated the effects of resveratrol (RSV) on this mRNA marker level, leading to cancer progression.Materials and Methods:U-87MG cell line was obtained from Pasteur Institute of Iran and treated with various concentrations of 0–160 μg/mL of RSV and at different time points (24, 48, and 72 h). To evaluate viability of U-87MG cells, standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed. Real-time polymerase chain reaction (RT-PCR) was used for comparative and quantitative assessment of human telomerase reverse transcriptase (hTERT) mRNA copy number versus control–untreated group.Results:The results of our investigation suggested that RSV effectively inhibited cell growth and caused cell death in dose-dependent (P < 0.05) and not in time-dependent manner (P > 0.05), in vitro. Interestingly, quantitative RT-PCR analysis demonstrated that at half inhibition concentration, RSV dramatically decreased mRNA expression of hTERT, the catalytic subunit of telomerase enzyme, which leads to prevention of cell division and tumor progression.Conclusion:With regard to downregulation of this immortalization-associated marker, RSV may potentially be used as a therapeutic agent against GBM.

Clinical and prognostic value of hTERT mRNA expression in patients with non-small-cell lung cancer.

Telomerase, undetectable in normal somatic cells, plays a critical role in carcinogenesis of the majority of human tumors including lung carcinoma. The aim of our study was to determine human telomerase reverse transcriptase (hTERT) mRNA expression in patients with non-small cell lung cancer (NSCLC) in order to estimate its usefulness as diagnostic and/or prognostic factor. hTERT expression was analyzed in a group of 12 females and 28 males with NSCLC using Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR method) in cancerous and non-cancerous lung tissues. Results were analyzed according to clinical data and one-, two-, and five-year survival rates. hTERT expression in the cancerous tissue was significantly higher than in the lung parenchyma free from neoplasm infiltration (p<0.05). There was no significant association between hTERT expression in the tumor tissue and age, gender, grading or clinical stage. A significant difference in hTERT expression between two types of histopathological patterns (adenocarcinoma and squamous cell carcinoma) was detected (p=0.01). No association between hTERT expression in NSCLC specimens and survival rates was found. hTERT mRNA detection by QRT-PCR in tumor and corresponding cancer-free tissues can be used as a diagnostic marker in patients with NSCLC, but seems not to be a prognostic factor.

Expression and significance of human telomerase reverse transcriptase mRNA in different subtypes of melanoma

Objective To detect the expression of human telomerase reverse transcriptase (hTERT) mRNA in the melanoma, and to analyze the relationship between the expression and subtypes and clinicopathological features of melanoma. Methods Expression of hTERT mRNA was detected by real-time quantitative PCR in 64 cases of melanoma and 30 cases of nevus. SPSS 17.0 software was used to analyze the relationship between hTERT mRNA expression and clinical pathological features of melanoma. Results The relative expression of hTERT mRNA in melanoma tissues was higher than that in nevus tissues [(52.43±5.42) vs (21.38±3.73), t = 4.72, P = 0.000]. The expression of hTERT mRNA in melanoma had no significant correlation with age, gender, ethnicity (all P > 0.05), but had relationship with subtypes, lymph node metastasis, Clark classification (all P 0.05). Conclusions The expression of hTERT mRNA in melanoma is high, especially in mucosal melanoma. hTERT may play an important role in the occurrence and development of melanoma. Key words: Melanoma; Mucous membrane; Human telomerase reverse transcriptase; Real-time fluorescence quantitative PCR

Enhancement of radiosensitivity of MCF-7 breast cancer cells subjected to X-ray or carbon-ion irradiations

The sensitivity of cancer cells to radiation therapy varies based on cell cycle phase. Here we evaluated the differences between X-ray and carbon-ion irradiation with respect to cellular radiosensitivity and cancer cycle arrest in the breast cancer cell line, MCF-7. The cell survival rate, cell cycle distribution and the presence of apoptosis were measured by clonogenic assay and flow cytometry. BRCA1 and p21 protein levels were analyzed by Western blot, and the levels of human telomerase reverse transcriptase (hTERT) mRNA expression and telomere length were detected with real-time polymerase chain reaction. The results show a significant dose-dependent effects on survival rate, apoptosis and protein levels in the carbon-ion group of MCF-7 cells. Decreased proliferation was not observed at 2 Gy X-ray irradiation. There were significant differences in cellular cycle arrest, apoptosis percentages and BRCA1 and p21 protein expression between X-ray and heavy-ion groups. The results indicated that increasing in BRCA1 and p21 expression, and attenuation of hTERT gene expression induced by heavy-ion irradiation in MCF-7 cells might relate to mechanism of cellular radiosensitivity in G2/M arrested phase.