hTERT mRNA Expression Levels Research Articles

Fabrication of magnetic niosomal platform for delivery of resveratrol: potential anticancer activity against human pancreatic cancer Capan-1 cell

Recently, the presence of different nanoparticles (NPs) has developed targeting drug delivery in treatment of cancer cell. Targeted drug delivery systems using NPs have shown great promise in improving the efficacy of intracellular uptake as well as local concentration of therapeutics with minimizing side effects. The current study planned to synthesized resveratrol-loaded magnetic niosomes nanoparticles (RSV-MNIONPs) and evaluate their cytotoxicity activity in pancreatic cancer cells. For this aim, magnetic nanoparticles (MNPs) were synthesized and loaded into niosomes (NIOs) by the thin film hydration technique and then characterized via DLS, FT-IR, TEM, SEM and VSM techniques. Moreover, the cytotoxic activity of the RSV-MNIONPs on the Capan-1 cells line was assessed by the MTT test. The distribution number of RSV-MNIONPs was gained about 80 nm and 95 nm with surface charge of − 14.0 mV by SEM and TEM analysis, respectively. RSV loading efficacy in NIOs was about 85%, and the drug releases pattern displayed a sustained discharge with a maximum amount about 35% and 40%, within 4 h in pH = 7.4 and pH = 5.8, respectively. The cytotoxicity of the RSV-MNIONPs in the presence of an external magnetic field is higher than that of the RSV, indicating enhanced cellular uptake in their encapsulated states. Furthermore, RSV loaded MNNPs were found to induce more cell cycle arrest at the G0/G1 checkpoint than free RSV. Compared with RSV-treated cells, the mRNA expression levels of BAX, Bcl2, FAS, P 53, Cyclin D and hTERT, were significantly changed in cells treated with RSV loaded MNNPs. The niosomes NPs approaches have been widely used to attain higher solubility, improved bioavailability, enhanced stability, and control delivery of RSV. Our formulation displayed antitumor activity and can be considered an appropriate carrier with a great potential for future usage in cancer therapy.

Angiotensin-(1–7) promotes mitochondrial translocation of human telomerase reverse transcriptase in HUVECs through the TOM20 complex

BackgroundAngiotensin (Ang) (1–7) is a vasodilator peptide that ameliorates microcirculation dysfunction, increases telomerase activity in cells, and exerts vasodilatory, anti-inflammatory, antioxidative stress, and antiapoptotic effects. Mitochondrial human telomerase reverse transcriptase (hTERT) plays an important role in the processes of antiapoptosis, antioxidative stress, and immortalization. This study aimed to investigate the effect of Ang(1–7) on the mitochondrial translocation of hTERT. MethodsAn in vitro model of lipopolysaccharide (LPS)-induced inflammation was established in human umbilical vein endothelial cells (HUVECs). Ang(1–7) was added to cells 30 min before LPS stimulation. The Ang(1–7)/Mas receptor antagonist A779 plus Ang(1–7) were added to the cells 30 min before LPS stimulation. The translocase outer membrane (TOM)20-overexpression HUVECs (HUVEC-TOM20OE), TOM20-knockdown HUVECs (HUVEC-TOM20KD), and the corresponding negative control cell lines were constructed by lentiviral transfection of HUVECs. Cells subjected to LPS stimulation alone, LPS plus Ang(1–7), LPS plus Ang(1–7) and A779, vehicle and no treatment were termed the LPS group, LPS + A group, LPS + A + A779 group, Con group and Neg group, respectively. Immunofluorescence staining was used to detect the distribution of hTERT in the nuclei and mitochondria of HUVECs and to locate TOM20, TOM40, and translocase inner membrane (TIM)23 in the mitochondria. The protein expression levels of total hTERT, mitochondrial hTERT, TOM20, TOM40, and TIM23 were measured by Western blot. The mRNA expression levels of hTERT, TOM20, TOM40, and TIM23 were assessed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). ResultshTERT colocalized with TOM40, TOM20 and TIM23 in the mitochondria. The mitochondrial hTERT protein level of the LPS + A group was significantly greater than that of the LPS group (P = 0.001), and the LPS group showed significantly increased expression of mitochondrial hTERT compared with that of the control group (P = 0.001). No significant difference in the level of total hTERT was observed between the LPS + A and LPS groups. The mitochondrial hTERT protein level of the LPS + A + A779 group was significantly lower than that of the LPS + A group (P = 0.021). The protein level of mitochondrial hTERT in HUVEC-TOM20KD treated with or without LPS alone or LPS + A was significantly decreased compared with the corresponding groups of control HUVECs (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.035; HUVEC-TOM20KD-LPS vs. HUVEC-LPS, P = 0.003; HUVEC-TOM20KD-LPS + A vs. HUVEC-LPS + A, P = 0.001), and treatment with Ang(1–7) did not restore the downregulation of mitochondrial hTERT in HUVEC-TOM20KD. HUVEC-TOM20OE showed a significantly increased level of mitochondrial hTERT (HUVEC-TOM20OE-Con vs. HUVEC-Con, P = 0.010), which was further elevated by Ang(1–7) stimulation (HUVEC-TOM20OE-LPS + A vs. HUVEC-TOM20OE-Con, P = 0.011). Lastly, the protein expression levels of TOM40 (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.007) and TIM23 (HUVEC-TOM20KD-Con vs. HUVEC-Con, P = 0.001) were significantly increased in HUVEC-TOM20KD in comparison to HUVECs. ConclusionsAng(1–7) effectively promoted mitochondrial translocation of hTERT in HUVECs via TOM20, indicating that hTERT may be transported to the mitochondria through the TOM20 complex. In addition, A779 could block the effects of Ang(1–7) in HUVECs.

A novel antisense oligonucleotide anchored on the intronic splicing enhancer of hTERT pre-mRNA inhibits telomerase activity and induces apoptosis in glioma cells

Alternative splicing of hTERT pre-mRNA is an important step in the regulation of telomerase activity, but the regulation mechanisms and functions remain unclear. RT-PCR analysis was used to detect hTERT splicing in glioma cell lines and brain tissues. TRAP assay was used to detect the telomerase activity. Then, we designed and synthesized 2'-O-methyl-RNA phosphorothioate AONs and transfected them into glioma cells to detect the changes in telomerase activity. MTT assay, plate colony formation assay, western blotting and Annexin V/PI assay were used to detect cell proliferation and apoptosis. At last, bioinformatics analyses were used to predict the expression and function of splicing protein SRSF2 in gliomas. hTERT splicing occurs both in glioma cell lines and glioma patients' tissues. The telomerase activity was related to the expression level of the full-length hTERT, rather than the total hTERT transcript level. AON-Ex726 was complementary to the sequence of the intronic splicing enhancer (ISE) in intron six, and significantly altered the splicing pattern of hTERT pre-mRNA, reducing the expression level of the full-length hTERT mRNA and increasing the expression level of the -β hTERT mRNA. After transfection with AON-Ex726, the level of apoptosis was increased, while telomerase activity and cell proliferation were significantly decreased. By bioinformatic predictions, we found the AON-Ex726 anchoring sequence in ISE overlaps the binding site of SRSF2 protein, which is up-regulated during the development of gliomas. Our findings provided new targets and important clues for the gene therapy of gliomas by regulating the alternative splicing pattern of hTERT pre-mRNA.

Human telomerase reverse transcriptase in papillary thyroid cancer: gene expression, effects of silencing and regulation by BET inhibitors in thyroid cancer cells.

Mutations in TERT promoter have been detected in the more aggressive papillary thyroid cancers (PTCs). To elucidate the role of TERT as an eligible molecular target in these tumors, the expression of hTERT was analyzed in a series of PTCs and the effects of both pharmacological and RNA-interference-induced hTERT silencing were investigated in two human PTC cell lines (K1 and BCPAP). The expression levels of hTERT mRNA and protein were evaluated by real-time PCR and western blot assays, respectively. Effects of hTERT silencing on PTC cell lines were analyzed by MTT, migration and western blot assays. Pharmacological inhibition of hTERT was performed using two bromodomain and extra-terminal (BET) inhibitors, JQ1 and I-BET762. hTERT expression results increased in 20 out of 48 PTCs, including tumors either positive or negative for the presence of hTERT promoter and/or BRAF mutations. In K1 and BCPAP cells, hTERT silencing determined a reduction in cell viability (~50% for K1 and ~70%, for BCPAP, vs control) and migration properties that were associated with a decrease of AKT phosphorylation and β-Catenin expression. Moreover, hTERT mRNA levels were down-regulated by two BET inhibitors, JQ1 and I-BET762, which at the same dosage (0.5 and 5 µM) reduced the growth of these thyroid cancer cells. These findings demonstrate that hTERT may represent an excellent therapeutic target in subgroups of aggressive PTCs.

1642P - Determination of biological behavior of solitary fibrous tumors

Background: Solitary fibrous tumor (SFT) is an uncommon neoplasm of pleural and extrapleural site of origin formed by cells with fibroblastic features. This tumor exhibits a spectrum of biological behavior and can occur at any age with the peak in the sixth and seventh decade of life. Most of these tumors grow slowly and their clinical behavior is mostly benign; however, up to 20 % of patients develop local recurrences and/or distant metastases. Methods: We performed a retrospective study of 54 cases of SFTs. We investigated morphologic characteristics, proliferation activity evaluated using Ki-67 immunostain and existence of NAB2-STAT6 fusion gene together with Ki-67, TPX2, and hTERT mRNA expression levels. The aim was to define relationships between proliferation activity and biological potential and progression of the disease. Recently, several alterations within the TERT gene have been detected in human tumors. The most frequent alteration in the TERT gene, somatic promoter mutation, has been described. We measured Ki-67, TPX2, and hTERT mRNA levels using quantitative real-time reverse transcription PCR (RQ-RT-PCR). Determination of hot spot promoter mutation in TERT was analyzed. Results: NAB2-STAT6 fusion transcript was found in 46/54 cases (85%) of amplifiable samples. The mRNA expression of Ki-67 correlated with local recurrences (p = 0.025) and biological behavior of the tumor (p = 0.0027), but did not correlate with the type of the NAB2/STAT6 fusion (0.24). The level of Ki-67 mRNA correlated with IHC established results (p = 0.02). The TPX2 mRNA expression did not correlate with local recurrences (p = 0.26) nor with biological behavior of the tumor (p = 0.062). The mRNA hTERT expression correlated well with biological behavior of the tumor (p < 0.0001). The majority of SFTs with benign behavior were without detectable expression of hTERT mRNA. A majority of the patients with hTERT expression had also somatic promoter mutation C228T or less frequently C250T. We observed a significant association between increased Ki-67 and hTERT mRNA levels and the SFTs with malignant potential. Conclusions: Detection of hTERT mRNA expression and its promoter mutation at routine practice might lead to a better estimation of a biological potential of SFTs. Legal entity responsible for the study: Ministry of Health, Czech Republic. Funding: Supported by the Project (Ministry of Health, Czech Republic) for conceptual development of research organization 00064203 (University Hospital Motol, Prague, Czech Republic). Disclosure: All authors have declared no conflicts of interest.

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.

The effects of thymoquinone and genistein treatment on telomerase activity, apoptosis, angiogenesis, and survival in thyroid cancer cell lines.

Thyroid cancers (TCs) are the most common endocrine malignancies. There were two problems with the current cancer chemotherapy: the ineffectiveness of treatment due to resistance to cancer cell, and the toxic effect on normal cells. This study was aimed to determine the effects of thymoquinone (TQ) and genistein (Gen) phytotherapeutics on telomerase activity, angiogenesis, and apoptosis in follicular and anaplastic thyroid cancer cells (TCCs). Cell viability, caspase-3 (CASP-3) activity, and messenger RNA (mRNA) expression levels of human telomerase reverse transcriptase (hTERT), phosphatase and tensin homolog (PTEN), nuclear factor-kappa B (NF-kB), cyclin-dependent kinase inhibitor 1 (p21), and vascular endothelial growth factor-A (VEGF-A) genes were analyzed. It was found that TQ and Gen treatment on TCCs caused a statistically significant decrease of cell viability, and mRNA expression levels of hTERT, VEGF-A, and NF-kB genes, but a statistically significant increase of PTEN and p21 mRNA expression levels. In addition, TQ and Gen treatment also caused a statistically significant increase active CASP-3 protein level in TCCs. Moreover, our results demonstrated that, when compared with follicular TCCs, anaplastic TCCs were more sensitive to the treatment of TQ and Gen. Based on these results, two agents can be good options as potential phytochemotherapeutics against TCCs.

Overexpression of MiR-138 Inhibits Cell Growth and Induces Caspase-mediated Apoptosis in Acute Promyelocytic Leukemia Cell Line.

Dysregulated expression of miRNAs can play a vital role in pathogenesis of leukemia. The shortened telomere length, and elevated telomerase activity in acute promyelocytic leukemia cells are mainly indicative of extensive proliferative activity. This study aimed to investigate the effect of overexpression of miR-138 on telomerase activity, and cell proliferation of acute promyelocytic leukemia NB4 cells. MiR-138 was overexpressed in NB4 cells using GFP hsa-miR-138-expressing lentiviruses. hTERT mRNA and protein expression levels were assessed by qRT-PCR and western blot analysis. For evaluation of apoptosis, annexin-V staining and activation of caspases were assessed using flow cytometry and western blot analysis, respectively. Our data demonstrate that overexpression of miR-138 attenuated the hTERT mRNA and protein expression levels. In addition, cell growth was inhibited, and malignant cells underwent caspase mediated-apoptosis in response to miR-138 overexpression. These findings suggest that loss of miR-138 expression may be associated with increased telomerase activity in NB4 cells. Therefore, strategies for up-regulation of miR-138 may result in inhibition of malignant cell growth, and provide a promising therapeutic approach for acute promyelocytic leukemia.

Correlation of hTERT Expression with Cervical Cytological Abnormalities and Human Papillomavirus Infection.

Telomerase Reverse Transcriptase (TERT) is the main catalytic sub-unit of telomerase, a reverse transcriptase enzyme. Telomerase expression is regulated at many levels, with numerous studies suggesting that up-regulation of human TERT gene (hTERT) at transcriptional level results in immortal cell phenotype associated with cancer. The aim of this study is to determine the correlation between hTERT expression and different cervical precursor lesions, as well as with cervical cancer in patients with confirmed Human papillomavirus (HPV) infection. The study included molecular analyzes on cervical samples from 214 women and matched Papanicolaou (Pap) test results. HPV detection and genotyping was performed by polymerase chain reaction (PCR) and genotyping. Quantitative real-time PCR (qRT-PCR) was performed using TaqMan probes and were calculated relative to the reference gene. Results showed significantly increased hTERT mRNA expression levels in high-grade and low-grade lesions compared to normal control samples (p<0.01) associated with 6.31 fold higher risk for developing ASC-US and 9.20 for LSIL. Strong correlation between HPV infection and hTERT expression in the high-grade lesions and cervical cancer was also observed. hTERT relative expression values showed 98% specificity and 100 % sensitivity as indicator of cervical lesions particularly for the ACS-H, HSIL and cervical cancer. In conclusion, hTERT expression correlate with the cytological grade of the cervical lesions and HPV infection and has a potential to be used as a diagnostic and prognostic marker.

Long-term persistent infection of HPV 16 E6 up-regulate SP1 and hTERT by inhibiting LKB1 in lung cancer cells.

HPV 16 E6 upregulates hTERT expression in lung cancer cells. However, the underlying molecular mechanism is unclear. In this paper, E6, LKB1, SP1, and hTERT mRNA expression levels were detected in brushing cells of patients with lung cancer (n = 106) and with benign lung disease (n = 68) by qRT-PCR. The mRNA expression levels of E6, SP1, and hTERT were significantly increased in the malignant group compared with the benign group (P < 0.01). Conversely, the mRNA expression level of LKB1 was significantly decreased in the malignant group (P < 0.01). Furthermore, the correlation between E6, Sp1, hTERT, and LKB1 was performed, our results indicated that E6, Sp1, and hTERT with positive, but LKB1 with negative correlation (P < 0.01). To investigate the potential relationship between these genes, using double directional genetic manipulation, we showed that overexpression of E6 in H1299 cells down-regulated LKB1 mRNA and protein expression but up-regulated SP1 and hTERT as well as the transcriptional activity of Sp1. In contrast, knockdown of E6 in A549 cells by short-interference RNAs (siRNAs) up-regulated LKB1 expression, but down-regulated SP1 and hTERT expression as well as Sp1 activity. LKB1 loss upregulated both SP1 and hTERT at the protein and mRNA level as well as SP1 activity. To verify that the role of E6 on hTERT was mediated by SP1, siRNA knockdown of SP1 was performed on both H1299 and A549 cell lines. Inhibition of SP1 downregulated hTERT expression. Our results indicate that HPV16 E6 indirectly upregulated the expression of hTERT by inhibition of LBK1 expression and upregulation of Sp1 expression, thus suggesting a HPV-LKB1-SP1-hTERT axis for the tumorigenesis of lung cancer. Our study also provides new evidence to support the critical role of SP1 and LKB1 in the pathogenesis of HPV-related lung cancer, and suggests novel therapeutic targets.

Role of GLTSCR2 in the regulation of telomerase activity and chromosome stability.

Telomerase is essential for regulating telomeres, and its activation is a critical step in cellular immortalization and tumorigenesis. The transcriptional activation of human telomerase reverse transcriptase (hTERT) is critical for telomerase expression. Although several transcriptional activators have been identified, factors responsible for enhancing the hTERT promoter remain to be fully elucidated. In the present study, the role of glioma tumor-suppressor candidate region gene2 (GLTSCR2) in telomerase regulation was analyzed. A doxycyclin-inducible green fluorescent protein (GFP)-tagged GLTSCR2-expressing adenovirus (Ad‑GLT/GFP) was used for the transduction of SK‑Hep‑1 and T98G cancer cells, and normal human umbilical vein endothelial cells. Changes in telomerase activity using telomere repeat amplification protocol assay were assessed, and the gene expression levels of hTERT were then examined. To investigate chromosome instability and senescence, Giemsa and β-galactosidase staining was performed. The results revealed that overexpression of GLTSCR2 significantly increased telomerase activity in the cancer and normal cell lines. This increase was consistent with increases in the protein and mRNA expression levels of hTERT. In luciferase assays, the hTERT promoter was activated by GLTSCR2. Knockdown of GLTSCR2 led to the downregulation of telomerase activity, abnormal nuclear morphology as a marker of chromosome instability, significant suppression of growth rate, alterations in cellular morphology and, eventually, cellular senescence. Taken together, the results of the present study suggested that GLTSCR2 is crucially involved in the positive regulation of telomerase and chromosome stability.

Expression of CLL Upregulated Gene1(CLLU) ,Telomere Reverse Transcriptase (hTERT) ,Zeta -Associated Protein Kinase -70 (ZAP-70) in patients with Chronic Lymphocytic Leukemia

Abstract Background Defining prognostic tool may help in B-CLL patient subgroups to benefit from early therapeutic intervention. ZAP-70 expression is one marker for IgVH mutational status. hTERT expression discriminated the Ig-unmutated from Ig-mutated B-CLL and may serve as a molecular prognostic marker. CLLU1 expression holds some prognostic information not offered by other biologic markers in B-CLL. This findings drove us forcibly to investigate their expression in B-CLL. Methods The current study was carried out on newly diagnosed 30 B-CLL patients before treatment. In Binet classification ,23% in stage A, 33.3% in stage B and 43.7% in stage C. lymphoadenopathy was in 76.7% , splenomegaly 66.7% ,hepatomegaly 20% of patients. PBMCs were isolated from B-CLL patients and tested by immunocytochemical staining technique for ZAP-70 expression and QRT-PCR for hTERT and CLLU1 expression using cDNA1 for CLLU1 Results ZAP-70 in mononuclear cells was positive in 30% of patients . ZAP-70 was significantly increased with Binet stage progression and . It was positively correlated with TLC and lymphocytic count and negatively correlated with the hemoglobin level and platelets count. The expression levels of hTERT mRNA and CLLU1 mRNA were +ive in all BCLL and -ive in normal cells. They were not correlated with Binet stage, hematological parameters ,ZAP-70 . The expression levels of hTERT and CLLU1 were higher in ZAP-70 positive but statistically non significant Conclusion ZAP-70 expression by Immunocytochemistry is a standardized, simple and inexpensive cost effective as a prognostic. hTERT and CLLU1 expression were novel prognostic markers in B-CLL that can be measured using a rapid and more sensitive method (QRT-PCR) accessible to routine laboratories.

The regulation of estradiol on growth dynamics of human LECs affected by increasing telomerase activity

Background Human LECs can express telomerase activity, which participates in the formation of cataract.It is reported that estrogen can increase the expression of telomerase activity in human endometrial cancer and breast cancer cells and play an important role in promoting proliferation and anti-apoptosis, but whether estrogen exerts its role on human LECs is still unclear. Objective This study aimed to investigate whether β-estradiol (β-E2) can increase the telomerase activity of human LECs and the influence of β-E2 on proliferation and apoptosis of human LECs. Method Human LECs line was cultured and passaged in vitro, and 1×10-6 mol/L β-E2 was added in the medium for 0, 6, 12, 24 and 48 hours, and reverse transcription PCR was used to determine the optimal time of the expression of human telomerase reverse transcriptase (hTERT) mRNA in the cells.Cultured cells were divided into five groups.The cells in the blank contol group were cultured in the routin medium.Ethanol of 0.1% was added in the solvent control group, and 1×10-8, 1×10-7 or 1×10-6 mol/L β-E2 was added in the medium in different contents of β-E2 groups, respectively.The relative expression level of hTERT mRNA in different groups was detected by reverse transcription PCR.Telomere repeat amplification protocol (TRAP)-ELISA was employed to determine the telomerase activity.The proliferative value of the cells was assayed by cell counting kit-8, and the apoptosis rate of the cells was examined by Hoechst33258 staining. Results The optimal time of β-E2 to rise the expression of hTERT mRNA (absorbance) was at 24 hours under the 1×10-6 mol/L.The relative expression levels of hTERT mRNA in the cells were 0.477±0.015, 0.712±0.013 and 0.914±0.031 in the 1×10-8, 1×10-7 and 1×10-6 mol/L β-E2 group, which were signifincatly higher than 0.428±0.010 in the blank control group and 0.426±0.010 in the solvent control group(all at P<0.05). The telomerase activity values (absorbance) were 0.711±0.015, 0.941±0.010 and 1.249±0.047 in the 1×10-8, 1×10-7 and 1×10-6 mol/L β-E2 group, which were higher than 0.535±0.013 in the blank control group and 0.543±0.013 in the solvent control group (all at P=0.000). The proliferantive values of the cells (absorbance) were significantly raised in the 1×10-8 mol/L β-E2 group compared with 1×10-7 and 1×10-6 mol/L β-E2 group (both at P=0.000), and no significnant difference was found in the proliferetive values between the blank control group and the solvent control group (P=0.718, 0.856). The apoptosis rates of the cells in the 1×10-8, 1×10-7 and 1×10-6 mol/L β-E2 group were lower than those in the the blank control group and the solvent control group (all at P=0.000), and there was no significant difference between the blank control group and the solvent control group (P=0.777). No obvious correlation was found between the HLECs preliferative values and hTERT mRNA expression levels or telomerase activity values (r=-0.299, P=0.278; r=-0.157, P=0.576). However, significantly negative correlations were seen between apoptosis rates and hTERT mRNA expression levels or telomerase activity values (r=-0.975, P=0.000; r=-0.981, P=0.000). Conclusions β-E2 can increase the activity of telomerase in human LECs, and high dose of β-E2 can inhibit apoptosis, but it dose not promote proliferation. Key words: Epithelial cells, lens; Estradiol; Telomerase; Cataract, age-related; Apoptosis; Proliferation; Cell, cultured; Cell line

Gastrointestinal stromal tumors - quantitative detection of the Ki-67, TPX2, TOP2A, and hTERT telomerase subunit mRNA levels to determine proliferation activity and a potential for aggressive biological behavior.

Gastrointestinal stromal tumors (GISTs) have an unpredictable biological potential ranging from benign to malignant. Molecular markers involved in the mechanisms of proliferation and cellular senescence may provide additional information about biological behavior of the tumor. The aim of the present study was to investigate Ki-67, TPX2, TOP2A and hTERT mRNA expression levels in specimens from patients with GISTs to define relationships between proliferation activity and biological potential and progression of the disease. We measured Ki-67, TPX2, TOP2A and hTERT mRNA levels using quantitative real-time reverse transcription PCR (RQ RT PCR). The highest Ki-67, TPX2, TOP2A and hTERT mRNA expression levels were found in the highly proliferative BLs (18 specimens), in comparison with GISTs (137 specimens) and LMSs (9 specimens). Patients with GISTs and adequate information about mitotic activity, tumor size and anatomical site (84 specimens) were divided into two groups - GISTs with benign (29 patients) and with malignant (55 patients) potential. We observed association between higher Ki-67, TPX2 and hTERT mRNA levels and the GISTs with malignant potential. Univariate analysis (57 patients with available follow-up information) of survival (Kaplan Meier curves method) revealed acorrelation between higher levels of TPX2, Ki-67 and hTERT markers and shorter event-free survival (EFS) or poorer overall survival (OS). The results demonstrate the importance of quantitative assessment of the proliferation activity in GISTs. Proliferation markers of Ki-67, TPX2, TOP2A and hTERT are suitable markers for detection the proliferation activity and telomerase activity of these tumors. Furthermore, the assessment of TPX2, Ki-67 and hTERT expression levels is appropriate for determination of malignant potential of GISTs.

Screening for Oral Cancer Using Electrochemical Telomerase Assay

AbstractElectrochemical telomerase assay (ECTA) developed by our group was evaluated in an oral cancer screening using exfoliated oral cells and tissues obtained from patients of oral cancer, mucosa associated disease, or healthy volunteers. Telomerase activity from ECTA is correlated with hTERT mRNA expression level using a real‐time PCR and was increasing in the following order: healthy volunteer group&lt;mucosa associated disease group&lt;oral cancer group. Sensitivity and specificity of ECTA were 88 % and 72 %, respectively when used 17 % of the threshold value based on the receiver operating characteristic curve in ECTA data.

Targeting Human Telomerase Reverse Transcriptase by a Simple siRNA Expression Cassette in HepG2 Cells.

Background:Human telomerase reverse transcriptase (hTERT) has become an ideal target for development of anticancer therapy. Small interfering RNAs (siRNAs) are very powerful reagents for gene silencing and show promise for cancer gene therapy. However, only a small number of siRNAs have been demonstrated to be effective. For gene therapy targeting hTERT, it is essential to develop a robust system to fully explore the power of siRNAs.Objectives:We explored a siRNA expression cassette (SEC) to screen highly effective RNAi-targeted sequences for gene therapy of hepatocellular carcinoma (HCC).Materials and Methods:An SEC was developed by flanking H1 and U6 promoters in opposite directions at the siRNA-encoding sequence. Eight SECs specific to hTERT were designed by overlap extension polymerase chain reaction (PCR) and transfected into HepG2 cells with calcium phosphate. The telomerase activity was determined by telomeric repeat amplification protocol (TRAP) silver staining and TRAP real-time PCR analysis. The mRNA and protein expression levels of hTERT were determined by reverse transcription (RT)-PCR and western blot, respectively. Cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell apoptosis was measured by the annexin-V/propidium iodide (PI) assay coupled with flow cytometry.Results:Eight hTERT-specific SECs (SEC-1-8) were successfully constructed. In comparison to that of the negative control SEC, the hTERT-specific SECs, especially, SEC-4, SEC-5, SEC-7 and SEC-8 significantly reduced the activity of hTERT in HepG2 cells at 48 hours after transfection. Moreover, the mRNA and protein expression levels of hTERT as well as the cell viability were significantly reduced by SECs. Knockdown of hTERT by SECs in HepG2 cells led to cell apoptosis.Conclusions:Our developed simple SEC was a powerful strategy for screening highly effective RNAi-targeted sequences and showed promise for gene therapy of HCC.

Use of hTERT and HPV E6/E7 mRNA RT-qPCR TaqMan assays in combination for diagnosing high-grade cervical lesions and malignant tumors.

Human papillomavirus (HPV) is a major cause of cervical cancer, which is the second most common cancer in women. HPV E6 initiates degradation of cellular tumor suppressor protein p53, induces human telomerase reverse transcriptase (hTERT) activity, and then leads to progressive cervical carcinogenesis. In this study, the CervicGen HPV RT-qDX assay (Optipharm, Osong, Republic of Korea), which detects 16 HPV high-risk subtypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 69), and the CervicGen hTERT RT-qDX assay (Optipharm) were evaluated using 545 ThinPrep (Hologic, Bedford, MA) Papanicolaou samples. The positivity for the HPV E6/E7 messenger RNA (mRNA) assay was 94.4%, 95.2%, 82.4%, 46.5%, 25.0%, and 1.1% in squamous cell carcinomas, high-grade squamous intraepithelial lesions (HSILs), atypical squamous cells--cannot exclude HSIL, low-grade squamous intraepithelial lesions, atypical squamous cells of undetermined significance, and normal cytology samples, respectively. Five cervical intraepithelial neoplasia grade 2+ samples were not detected by the HPV E6/E7 mRNA assay, but they exhibited positive signals in the hTERT mRNA assay. Notably, the hTERT mRNA expression level was increased in high-grade cervical lesions but was very low in all 288 normal samples. These data suggest that the combination of HPV E6/E7 and hTERT mRNA expression levels could be used in a complementary manner in diagnosing high-grade cervical lesions and malignant tumors and might be useful as a predictive marker in monitoring low-grade cervical lesions.

Molecular Diagnosis of Hepatocellular Carcinoma in Egypt

Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It is a prevalent cancer that often develops in patients with chronic hepatitis and cirrhosis in association with hepatitis B or C virus infection. Diagnostic modalities such as tumor markers, ultrasonography and CT have contributed to its earlier diagnosis but remain not sensitive enough. Telomeres are specializes structures at the ends of eukaryotic chromosomes and lead to their stabilization. Telomerase is a ribonucleoprotein enzyme responsible for replication of telomeres at chromosomal ends in most eukaryotes.Its RNA subunit provides the template for addition of (GGTTAG) repeats to chromosome ends .Human telomerase mRNA (hTERT- mRNA)has been identified in many cancers and claimed to be reactivated in HCC.Aim: to investigate Human telomerase mRNA (hTERT- mRNA) in peripheral blood of hepatocellular carcinoma (HCC) and chronic liver diseases (CLD) patients,To correlate the level of Human telomerase mRNA (hTERT- mRNA) with alpha feto –protein ( AFP),the traditional serological marker for HCC.Patients and methods:The study was conducted on 60 patients selected from the National Liver Institute clinics and inpatients hepatology department. The patients were divided into group I (30 patients with CLD) and group II (30 patients diagnosed to have HCC). In addition to group III that comprised 20 apparantly healthy volunteers .All selected individuals were subjected to history taking ,thorough clinical examination, abdominal ultrasonography and routine laboratory investigations as liver function tests ,CBC, hepatitis viral markers , serum AFP and quantitative detection of m-RNA expression encoding for telomerase catalytic subunit hTERT by real time PCR measurement using 7500 Real Time PCR System(Applied Biosystems).Results: A significant elevation in AFP level in HCC group when compared to CLD patients group or to control group (p < 0.01).The mean level of hTERT m-RNA expression in HCC patients group was significantly higher than both CLD patients group and controls (p<0.01). An hTERT m-RNA expression cut off level of 81.5 copies /ml showed prediction of HCC 100% sensitivity,95 % specificity, accuracy 99.6% ,standard error (SE) was 0.003 and confidence interval (CI) was (0.99- Conclusion: Real time assay of hTERT m-RNA in patients with CLD could be used as a satisfactory molecular marker for diagnosis of HCC. hTERT m-RNA is thought to be superior to AFP for early detection of HCC.

The effect of silencing human telomerase reverse transcriptase by RNA interference on U87 glioma cell proliferation and apoptosis

Objective To observe the effect of human telomerase reverse transcriptase (hTERT) on U87 glioma cell proliferation and apoptosis.Methods The small interfering RNA (siRNA) expression plasmid targeting hTERT was constructed and transfected into U87 glioma cells.The expression level of hTERT protein and mRNA was detected by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) respectively.The U87 glioma cell proliferation and apoptosis were analyzed by methyl thiazol tetrazolium (MTT) assay and flow cytometry,respectively.Results Compared with the blank control group and the negative control group,the expression level of hTERT protein in each siRNA group was significantly reduced (P ＜ 0.05).The relative expression level of hTERT mRNA in each siRNA group was 0.79 ±0.11,0.58 ±0.15 and 0.39 ±0.16 respectively,which was significantly lower than in the negative control group (P ＜ 0.05).As compared with the blank control group and the negative control group,the proliferation of U87 glioma cells after transfection was significantly inhibited and the strongest inhibitory effect was observed on the third day.The apoptosis rate of U87 glioma cells of in each siRNA group which was transfected after 48 h was (14.31 ± 0.93) ％,(17.57 ± 0.61) ％ and (22.68 ± 1.04) ％ respectively,which was significantly higher than in the blank control group and the negative control group (P ＜0.05).Conclusion The silence of hTERT gene after siRNA targeting hTERT being transfected into U87 glioma cells can significantly inhibit cell proliferation and induce cell apoptosis. Key words: RNA interference; Human telomerase reverse transcriptase; Glioma; Cell proliferation

Epigenetic mechanisms regulate placental c-myc and hTERT in normal and pathological pregnancies; c-myc as a novel fetal DNA epigenetic marker for pre-eclampsia.

Placental development is known for its resemblance with tumor development, such as in the expression of oncogenes (c-myc) and telomerase (hTERT). The expression of c-myc and hTERT is up-regulated during early pregnancy and gestational trophoblastic diseases (GTDs). To determine the role of DNA methylation [via methylation-sensitive high resolution melting (MS-HRM)] and histone modifications [via chromatin immunoprecipitation (ChIP assay)] in regulating the differential expression of c-myc and hTERT during normal gestation and their dysregulation during placental disorders, we obtained placental samples from 135 pregnant women, in five groups: normal first, second and third trimester (n = 30 each), pre-eclamptic pregnancy (n = 30) and molar pregnancy (n = 15). Two placental cell lines (JEG-3 and HTR-8/SVneo) and isolated first-trimester cytotrophoblasts were also studied. Quantitative RT-PCR revealed decreased mRNA expression levels of c-myc and hTERT, which were associated with a higher level of H3K9me3 (1.5-fold, P < 0.05) and H3K27me3 (1.9-fold, P < 0.05), respectively, in third-trimester placental villi versus first-trimester villi. A significantly lower level of H3K27me3 in molar placenta was associated with a higher mRNA expression of c-myc and hTERT. The development of pre-eclampsia (PE) was associated with increased methylation (P < 0.001) and H3K27me3 (P < 0.01) at the c-myc promoter and reduced H3K9me3 (P < 0.01) and H3K27me3 (P < 0.05) at the hTERT promoter. Further, mRNA expression of c-myc and hTERT was strongly correlated in molar villi (r = 0.88, P < 0.01) and JEG-3 cells (r = 0.99, P < 0.02). Moreover, on the basis of methylation data, we demonstrate the potential of c-myc as a fetal DNA epigenetic marker for pre-eclamptic pregnancies. Thus we suggest a role for epigenetic mechanisms in regulating differential expression of c-myc and hTERT during placental development and use of the c-myc promoter region as a potential fetal DNA marker in the case of PE.