Telomerase Gene Research Articles

Realtime qRT-PCR Expression of P53, KRAS, and human Telomerase genes in circulating tumor cells, as potential biomarkers for early detection of sporadic colorectal cancer

Objectives: The objectives of present study were to investigate the potential use of mRNA of P53, KRAS, and human telomerase genes in circulating tumor cells as biomarkers for early detection of sporadic cases of colorectal cancer. Background: Inspite of colorectal cancer is a curable disease if detected early; it is still the third leading cause of death from cancer. There is an ultimate need for implementing an accurate and noninvasive screening method. The recent advances in molecular genetics have showed that the genes of P53, KRAS and human telomerase play major roles in development of colorectal cancer. Therefore the study of their expression in blood samples might be used as a mean for early detection of sporadic colorectal carcinoma cases. Methods: A case control pilot study in which 20 patients with non-metastatic colorectal carcinoma in Duke’s stages (A – C, TNM from 0 – III) and 20 healthy controls were enrolled. Expression of studied genes and the housekeeping gene Glyceraldehyde-3 phosphate dehydrogenase were determined using quantitative reverse transcriptase polymerase chain reaction. The study was performed in duplicate taking 1µg of total RNA from each sample. Results: The results showed significant overexpression of mRNAs of studies genes in samples taken from cases of proved colorectal carcinoma compared to that of controls group with a statistically significant P- value of ​

Clinical study to look at treating degenerative disc disease by telomerase gene therapy

Clinical study to look at treating degenerative disc disease by telomerase gene therapy

Fanconi Anemia germline variants as susceptibility factors in aplastic anemia, MDS and AML.

Using next generation sequencing we have systematically analyzed a large cohort of 489 patients with bone marrow failure (BMF), including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), aplastic anemia (AA), and related conditions for the presence of germline (GL) alterations in Fanconi Anemia (FA) and telomerase genes. We have detected an increased frequency of heterozygous FA gene mutations in MDS and to lesser degree in AML suggesting that the presence of one normal allele may not be completely protective and indeed heterozygous FA lesions may have a long latency period before hematologic manifestation. In contrast, GL telomerase gene mutations were not associated with increased disease risk. When compared to large control cohorts, we have not detected an increased frequency of damaging variants among telomerase complex genes, including those previously believed to be involved in the pathogenesis of AA. Our results may suggest that while low penetrance and delayed disease onset can confound identification of genetic predisposition factors, GL FA alterations can be also associated with MDS.

Regulation of human and mouse telomerase genes by genomic contexts and transcription factors during embryonic stem cell differentiation

Differential regulation of telomerase reverse transcriptase (TERT) genes contribute to distinct aging and tumorigenic processes in humans and mice. To study TERT regulation, we generated mouse embryonic stem cell (ESC) lines containing single-copy bacterial artificial chromosome (BAC) reporters, covering hTERT and mTERT genes and their neighboring loci, via recombinase-mediated BAC targeting. ESC lines with chimeric BACs, in which two TERT promoters were swapped, were also generated. Using these chromatinized BACs, we showed that hTERT silencing during differentiation to embryoid bodies (EBs) and to fibroblast-like cells was driven by the human-specific genomic context and accompanied by increases of repressive epigenetic marks, H3K9me3 and H3K27me3, near its promoter. Conversely, the mouse genomic context did not repress TERT transcription until late during differentiation. The hTERT promoter was more active than its mouse counterpart when compared in the same genomic contexts. Mutations of E-box and E2F consensus sites at the promoter had little effect on hTERT transcription in ESCs. However, the mutant promoters were rapidly silenced upon EB differentiation, indicating that transcription factors (TFs) bound to these sites were critical in maintaining hTERT transcription during differentiation. Together, our study revealed a dynamic hTERT regulation by chromatin environment and promoter-bound TFs during ESC differentiation.

Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells.

The use of mesenchymal stem cells (MSCs) for cell therapy and regenerative medicine has received widespread attention over the past few years, but their application can be complicated by factors such as reduction in proliferation potential, the senescent tendency of the MSCs upon expansion and their age-dependent decline in number and function. It was shown that all the mentioned features were accompanied by a reduction in telomerase activity and telomere shortening. Furthermore, the role of epigenetic changes in aging, especially changes in promoter methylation, was reported. In this study, MSCs were isolated from the adipose tissue with enzymatic digestion. In addition, immunocytochemistry staining and flow cytometric analysis were performed to investigate the cell-surface markers. In addition, alizarin red-S, sudan III, toluidine blue, and cresyl violet staining were performed to evaluate the multi-lineage differentiation of hADSCs. In order to improve the effective application of MSCs, these cells were treated with 1.5 × 10−8 and 2.99 × 10−10 M of ZnSO4 for 48 hours. The length of the absolute telomere, human telomerase reverse transcriptase (hTERT) gene expression, telomerase activity, the investigation of methylation status of the hTERT gene promoter and the percentage of senescent cells were analyzed with quantitative real-time PCR, PCR-ELISA TRAP assay, methylation specific PCR (MSP), and beta-galactosidase (SA-β-gal) staining, respectively. The results showed that the telomere length, the hTERT gene expression, and the telomerase activity had significantly increased. In addition, the percentage of senescent cells had significantly decreased and changes in the methylation status of the CpG islands in the hTERT promoter region under treatment with ZnSO4 were seen. In conclusion, it seems that ZnSO4 as a proper antioxidant could improve the aging-related features due to lengthening of the telomeres, increasing the telomerase gene expression, telomerase activity, decreasing aging, and changing the methylation status of hTERT promoter; it could potentially beneficial for enhancing the application of aged-MSCs.

Snail1 transcription factor controls telomere transcription and integrity.

Besides controlling epithelial-to-mesenchymal transition (EMT) and cell invasion, the Snail1 transcriptional factor also provides cells with cancer stem cell features. Since telomere maintenance is essential for stemness, we have examined the control of telomere integrity by Snail1. Fluorescence in situ hybridization (FISH) analysis indicates that Snail1-depleted mouse mesenchymal stem cells (MSC) have both a dramatic increase of telomere alterations and shorter telomeres. Remarkably, Snail1-deficient MSC present higher levels of both telomerase activity and the long non-coding RNA called telomeric repeat-containing RNA (TERRA), an RNA that controls telomere integrity. Accordingly, Snail1 expression downregulates expression of the telomerase gene (TERT) as well as of TERRA 2q, 11q and 18q. TERRA and TERT are transiently downregulated during TGFβ-induced EMT in NMuMG cells, correlating with Snail1 expression. Global transcriptome analysis indicates that ectopic expression of TERRA affects the transcription of some genes induced during EMT, such as fibronectin, whereas that of TERT does not modify those genes. We propose that Snail1 repression of TERRA is required not only for telomere maintenance but also for the expression of a subset of mesenchymal genes.

Two-step role for mutant TERT promoters

Cancer Telomeres preserve genomic stability by preventing chromosomal fusions. The recent discovery that human tumors harbor mutations in the promoter region of the telomerase gene ( TERT ) produced a flurry of research aimed at elucidating the role of these mutations in cancer development. Chiba et al. present data that reconcile many of the conflicting results reported to date (see the Perspective by Shay). In human melanoma samples and a fibroblast model, TERT promoter mutations acted in two steps. First, the mutations allowed cells to proliferate with short telomeres. This fueled genomic instability and up-regulation of telomerase expression, leading to uncontrolled cell proliferation. Science , this issue p. [1416][1]; see also p. [1358][2] [1]: /lookup/doi/10.1126/science.aao0535 [2]: /lookup/doi/10.1126/science.aao6963

Assessment on application value of combination of TCT, HPV, c-MYC and hTERC genes to screen cervical cancer

Objective To investigate the application value of combination of thinprep cytologic test (TCT), human papilloma virus (HPV), c-MYC and human chromosome telomerase gene (hTERC) genes to screen cervical cancer. Methods A total of 230 cases of the study objects was detected with TCT and HP, respectively. Amplification of c-MYC and hTERC genes was tested with fluorescence in situ hybridization (FISH) method. The histopathological results were the gold standard, and the sensitivity, specificity and accuracy of cervical intraepithelial neoplasia (CIN) Ⅱ/Ⅲ and squamous cell carcinoma (SCC) were compared with the four combined detection. Results Of 230 screened patients, there were 124 cases of TCT positive, 155 cases of HPV positive, c-MYC gene amplified in 118 cases, and hTERC gene amplified in 128 cases. When TCT, HPV, c-MYC, and hTERC were used alone, the highest sensitivity was HPV (84.5%), the highest specificity was c-MYC (97.6%), and the highest accuracy was hTERC (85.2%). When the three indexes were used in coordination with each other, the sensitivity and accuracy of TCT+ HPV+ hTERC were the highest (98.6% and 90.9%), and the specificity of TCT+ c-MYC+ hTERC was the highest (79.3%). When the four indexes were used together, the sensitivity was 98.6%, the specificity was 72.0%, and the accuracy was 89.1%. Conclusions Combined examination can improve the sensitivity and accuracy of screening cervical lesions, and the three combination of TCT+ HPV+ hTERC had the best effect, and c-MYC gene detection had the highest specificity. Key words: Biopsy; Papillomaviridae; Genes, myc; Telomerase; Uterine cervical neoplasms/DI

Effects of Pyridostigmine bromide on SH-SY5Y cells: An in vitro neuroblastoma neurotoxicity model

Pyridostigmine bromide (PB) is a reversible acetylcholinesterase (AChE) inhibitor and the first-choice for the treatment of symptoms associated with myasthenia gravis and other neuromuscular junction disorders. However, evidence suggested that PB could be associated with the Gulf War Illness characterised by the presence of fatigue, headaches, cognitive dysfunction, and musculoskeletal respiratory and gastrointestinal disturbances. Given that a potential neurotoxic effect of PB has not yet been completely elucidated, the present investigation used neural SH-SY5Y cells to evaluate the effect of PB on the cellular viability, cell apoptosis, modulation of the cell cycle, oxidative stress, and genotoxicity variables, which indicate neurodegeneration. As expected, a PB concentration curve based on the therapeutic dose of the drug showed an inhibition of the AChE activity. However, this effect was transient and did not involve differential AChE gene regulation by PB. These results confirmed that undifferentiated SH-SY5Y cells can be used as a cholinergic in vitro model. In general, PB did not trigger oxidative stress, and at a slightly higher PB concentration (80ng/mL), higher levels of protein carbonylation and DNA damage were detected, as determined by the marker 8-deoxyguanosine. The PB genotoxic effects at 80ng/mL were confirmed by the upregulation of the p53 and DNA methyltransferase 1 (DNMT1) genes, which are associated with cellular DNA repair. PB at 40ng/mL, which is the minimal therapeutic dose, led to higher cell proliferation and mitochondrial activity compared with the control group. The effects of PB were corroborated by the upregulation of the telomerase gene. In summary, despite the methodological constrains related to the in vitro protocols, our results suggested that exposure of neural cells to PB, without other chemical and physical stressors did not cause extensive toxicity or indicate any neurodegeneration patterns.

Mutations in the promoter of the telomerase gene TERT contribute to tumorigenesis by a two-step mechanism.

TERT promoter mutations (TPMs) are the most common noncoding mutations in cancer. The timing and consequences of TPMs have not been fully established. Here, we show that TPMs acquired at the transition from benign nevus to malignant melanoma do not support telomere maintenance. In vitro experiments revealed that TPMs do not prevent telomere attrition, resulting in cells with critically short and unprotected telomeres. Immortalization by TPMs requires a gradual up-regulation of telomerase, coinciding with telomere fusions. These data suggest that TPMs contribute to tumorigenesis by promoting immortalization and genomic instability in two phases. In an initial phase, TPMs do not prevent bulk telomere shortening but extend cellular life span by healing the shortest telomeres. In the second phase, the critically short telomeres lead to genome instability and telomerase is further up-regulated to sustain cell proliferation.

Telomere biology and telomerase mutations in cirrhotic patients with hepatocellular carcinoma.

Telomeres are repetitive DNA sequences at linear chromosome termini, protecting chromosomes against end-to-end fusion and damage, providing chromosomal stability. Telomeres shorten with mitotic cellular division, but are maintained in cells with high proliferative capacity by telomerase. Loss-of-function mutations in telomere-maintenance genes are genetic risk factors for cirrhosis development in humans and murine models. Telomerase deficiency provokes accelerated telomere shortening and dysfunction, facilitating genomic instability and oncogenesis. Here we examined whether telomerase mutations and telomere shortening were associated with hepatocellular carcinoma (HCC) secondary to cirrhosis. Telomere length of peripheral blood leukocytes was measured by Southern blot and qPCR in 120 patients with HCC associated with cirrhosis and 261 healthy subjects. HCC patients were screened for telomerase gene variants (in TERT and TERC) by Sanger sequencing. Age-adjusted telomere length was comparable between HCC patients and healthy subjects by both Southern blot and qPCR. Four non-synonymous TERT heterozygous variants were identified in four unrelated patients, resulting in a significantly higher mutation carrier frequency (3.3%) in patients as compared to controls (p = 0.02). Three of the four variants (T726M, A1062T, and V1090M) were previously observed in patients with other telomere diseases (severe aplastic anemia, acute myeloid leukemia, and cirrhosis). A novel TERT variant, A243V, was identified in a 65-year-old male with advanced HCC and cirrhosis secondary to chronic hepatitis C virus (HCV) and alcohol ingestion, but direct assay measurements in vitro did not detect modulation of telomerase enzymatic activity or processivity. In summary, constitutional variants resulting in amino acid changes in the telomerase reverse transcriptase were found in a small proportion of patients with cirrhosis-associated HCC.

Abstract 4881: Dissecting telomere maintenance mechanisms in neuroblastoma

Abstract Background. Telomere maintenance is a major cancer hallmark and has recently attracted attention as an oncogenic mechanism in the pediatric malignancy, neuroblastoma (NBL), due to detection of frequent structural rearrangements near the telomerase (TERT) gene. NBLs display substantial clinical and molecular heterogeneity; tumors with MYCN amplification and TERT-associated rearrangements define separate groups of high-risk patients with active TERT. ATRX mutations are frequent in non-TERT active high-risk tumors, indicating use of alternative lengthening of telomeres (ALT) as a maintenance mechanism. Furthermore, the 4S low-risk NBLs display an elevated rate of spontaneous regression and overall good prognosis; it has been hypothesized that lacking a telomere maintenance mechanism (“telomere crisis”) may be behind this phenomenon. Methods. To gain mechanistic insight behind these phenomena, we introduce an integrative analysis of a cohort comprising 104 high-risk (32 MYCN-amplified) and 23 4S tumors with matched blood/tumor whole genome sequencing, CpG methylation, and RNA-seq data from the TARGET consortium. Here, we preformed structural variant (SV), allelic specific expression (ASE), and differential CpG methylation analyses. Additionally, we introduce a novel approach to measure relative telomere DNA abundance from short-read WGS measured as the ratio between tumor and blood telomeric reads (containing canonical repeats TTAGGG/CCCTAA) per million (TBrpm). Results. We identified 26 tumors harboring TERT rearrangements (25% of all high risk tumors) and 3 tumors with ATRX mutations. We define 4 groups of high-risk NBL with different telomere maintenance mechanisms: 1) Tumors with MYCN amplification or elevated MYC/N activity, TERT gene body hyper-methylation (β-value≅0.8), and expression of biallelic TERT; 2) Tumors harboring TERT associated SVs, hemi-methylation (β-value≅0.5), and expression of mono-allelic TERT, 3) Tumors without TERT expression, including ATRX mutants with hypo-methylated TERT (β-value≅0.3), 4) tumors with abnormally high telomeric DNA abundance (TBrpm > 1.5), hypo-methylation (β-value≅0.3), and no expression of TERT. Intriguingly, while groups 1-3 show telomeric loss (TBrpm ≅ 0.7-0.9), 4S tumors show conservation of telomere abundance between tumor and blood (TBrpm ≅ 1); hence no indication of telomere crisis, perhaps due to early timing of biopsy at diagnosis. Conclusion. We have used ASE status and gene body methylation of TERT in order to understand and validate mechanisms underlying TERT activation; we extend MYCN-driven TERT activation to tumors with high MYC/MYCN activity in the absence of MYCN amplification. Our telomere analysis suggests that different ALT mechanisms might take place in NBLs. Defining the mechanism of 4S NBL spontaneous regression requires further investigation. Citation Format: Gonzalo Lopez, Karina Conkrite, Kendra Hong, Jo Lynne Harenza, John M. Maris, Sharon Diskin. Dissecting telomere maintenance mechanisms in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4881. doi:10.1158/1538-7445.AM2017-4881

Establishment of a new conditionally immortalized human skeletal muscle microvascular endothelial cell line.

In skeletal muscle, the capillaries have tight junctions (TJs) that are structurally similar to those in the blood-brain barrier (BBB) and blood-nerve barrier (BNB). Although many findings have been clarified in the territory of BBB and BNB, few have so far examined the TJs of capillaries in the skeletal muscle. In addition, no in vitro human skeletal muscle microvasculature models have been reported thus far. We newly established a new human skeletal muscle microvascular endothelial cell (HSMMEC) line. HSMMECs were isolated from human skeletal muscle and were infected with retroviruses harboring temperature-sensitive SV40 T antigen and telomerase genes. This cell line, termed TSM15, showed a spindle fiber-shaped morphology, an immunoreactivity to anti-factor VIII and anti-VE-cadherin antibodies, and a temperature-sensitive growth. TSM15 cells grew stably for more than 40 passages when they were cultured at 33°C, thereby retaining their spindle fiber-shaped morphology and contact inhibition at confluence. The cells expressed tight junctional molecules such as claudin-5, occludin, and zonula occludens-1, as well as transporters such as a glucose transporter 1. The transendothelial electrical resistance of TSM15 was as high as those of the human brain microvascular endothelial cell line. This novel cell line might facilitate the analyses of the pathophysiology of inflammatory myopathy, such as dermatomyositis, and can improve our understanding of the physiological and biochemical properties of the microvasculature in human skeletal muscle.

Kaempferol increases apoptosis in human cervical cancer HeLa cells via PI3K/AKT and telomerase pathways.

Cervical cancer is one of the most frequent cancers in women worldwide. Defects in the apoptotic pathways are responsible for both the disease pathogenesis and its therapy resistance. It is thus a good candidate for treatment by pro-apoptotic agents. Kaempferol as a flavonoid has antioxidant and anti-tumor properties. Kaempferol has been shown to induce apoptosis and cell death in cancer cells. However, due to the problems in the treatment of cervical cancer, this study is designed to investigate the molecular mechanism by which kaempferol suppresses the growth of cervical cancer HeLa cell as compared with HFF cells (normal cells). Cells treated with kaempferol (12-100μM) and 5-FU (1-10μM), as the positive control, up to 72h. Cell viability was determined by MTT assay and real time PCR was used to investigate apoptosis and telomerase genes expression. The results showed that kaempferol decreased cell viability as concentration- and time-dependently. IC50 values were 10.48μM for HeLa and 707.00μM for HFF cells, as compared with 1.40μM and 16.38μM for 5-FU after 72h treatment, respectively. Also, kaempferol induced cellular apoptosis and aging through down-regulating the PI3K/AKT and hTERT pathways. This study suggests that kaempferol may be a useful adjuvant therapeutic agent in the treatment of cervical cancer.

Telomere length in patients with pulmonary fibrosis associated with chronic lung allograft dysfunction and post–lung transplantation survival

Prior studies have shown that patients with pulmonary fibrosis with mutations in the telomerase genes have a high rate of certain complications after lung transplantation. However, few studies have investigated clinical outcomes based on leukocyte telomere length. We conducted an observational cohort study of all patients with pulmonary fibrosis who underwent lung transplantation at a single center between January 1, 2007, and December 31, 2014. Leukocyte telomere length was measured from a blood sample collected before lung transplantation, and subjects were stratified into 2 groups (telomere length <10th percentile vs ≥10th percentile). Primary outcome was post-lung transplant survival. Secondary outcomes included incidence of allograft dysfunction, non-pulmonary organ dysfunction, and infection. Approximately 32% of subjects had a telomere length <10th percentile. Telomere length <10th percentile was independently associated with worse survival (hazard ratio 10.9, 95% confidence interval 2.7-44.8, p = 0.001). Telomere length <10th percentile was also independently associated with a shorter time to onset of chronic lung allograft dysfunction (hazard ratio 6.3, 95% confidence interval 2.0-20.0, p = 0.002). Grade 3 primary graft dysfunction occurred more frequently in the <10th percentile group compared with the ≥10th percentile group (28% vs 7%; p = 0.034). There was no difference between the 2 groups in incidence of acute cellular rejection, cytopenias, infection, or renal dysfunction. Telomere length <10th percentile was associated with worse survival and shorter time to onset of chronic lung allograft dysfunction and thus represents a biomarker that may aid in risk stratification of patients with pulmonary fibrosis before lung transplantation.

Defects in lymphocyte telomere homeostasis contribute to cellular immune phenotype in patients with cartilage-hair hypoplasia

Mutations in the long noncoding RNA RNase component of the mitochondrial RNA processing endoribonuclease (RMRP) give rise to the autosomal recessive condition cartilage-hair hypoplasia (CHH). The CHH disease phenotype has some overlap with dyskeratosis congenita, a well-known "telomere disorder." RMRP binds the telomerase reverse transcriptase (catalytic subunit) in some cell lines, raising the possibility that RMRP might play a role in telomere biology. We sought to determine whether a telomere phenotype is present in immune cells from patients with CHH and explore mechanisms underlying these observations. We assessed proliferative capacity and telomere length using flow-fluorescence in situ hybridization (in situ hybridization and flow cytometry) of primary lymphocytes from patients with CHH, carrier relatives, and control subjects. The role of telomerase holoenzyme components in gene expression and activity were assessed by using quantitative PCR and the telomere repeat amplification protocol from PBMCs and enriched lymphocyte cultures. Lymphocyte cultures from patients with CHH display growth defects invitro, which is consistent with an immune deficiency cellular phenotype. Here we show that telomere length and telomerase activity are impaired in primary lymphocyte subsets from patients with CHH. Notably, telomerase activity is affected in a gene dose-dependent manner when comparing heterozygote RMRP carriers with patients with CHH. Telomerase deficiency in patients with CHH is not mediated by abnormal telomerase gene transcript levels relative to those of endogenous genes. These findings suggest that telomere deficiency is implicated in the CHH disease phenotype through an as yet unidentified mechanism.

Establishment and Characterization of Vaginal Tissue Primary Culture: Feasibility of Cell Therapy for Mayer-Rokitansky-Kuster-Hauser Syndrome (MRKHS) Patient Treatment

Title: Establishment and Characterization of Vaginal Tissue Primary Culture: Feasibility of Cell Therapy Usage in Mayer- Rokitansky-Kuster-Hauser Syndrome (MRKHS) Patient´s Treatment. Background: The uterine and vaginal agenesis, Mayer- Rokitansky-Küster-Hauser Syndrome (SMRKH), is a congenital malformation that implies in the impairment of sexual activity and reproductive life. The first line therapy is nonsurgical (dilatation method) but surgical methods may be necessary to create a functional neovagina. In the surgical procedure, various types of graft materials have been used to line the neovagina, and recently, alternative grafts have been studied to minimize adverse effects. Our aim was to establish vaginal primary cultures in order to prove the feasibility of cell therapy for the reconstruction of the vaginal lining. Methods and findings: Nine small biopsies were processed according to explant technique in order to isolate vaginal cells (VC). Different cell types were obtained, including fibroblast and epithelial cells, according to optical microscopy, flow cytometry and immunofluorescence assessment. These cells could not differentiate into mesenchymal lineages (adipogenic, chondrogenic and osteogenic) but are able to form tissue-like cell aggregates. The expression of telomeres and telomerase genes were similar to the endometrial fibroblast. Conclusion: We are able to obtain and characterize vaginaderived cells in primary cultures. The isolated cells seem to be good candidates to cell therapy usage in MRKHS as they have phenotypic and physiological characteristics suitable to the reconstruction of vaginal lining.

Evaluation of Energy Balance on Human Telomerase Reverse Transcriptase (hTERT) Alternative Splicing by Semi-quantitative RT-PCR in Human Umbilical Vein Endothelial Cells.

Background:Decreased high-energy phosphate level is involved in endothelial cell injury and dysfunction. Reduced telomerase activity in endothelial cells in parallel with reduced energy levels might be due to altered direction of alternative splicing machine as a complication of depleted energy during the process of atherosclerosis.Materials and Methods:Isolated human umbilical vein endothelial cells (HUVECs) were treated for 24 hours by oligomycine (OM) and 2-deoxy glucose (2-DG). After 24 hours, the effect of energy depletion on telomerase splicing pattern was evaluated using RT-PCR. Indeed, in both treated and untargeted cells, nitric oxide (NO) and von Willebrand factor (vWF) were measured.Results:ATP was depleted in treated cells by 43.9% compared with control group. We observed a slight decrease in NO levels (P = 0.09) and vWF (P = 0.395) in the setting of 49.36% ATP depletion. In both groups, no telomerase gene expression was seen. Telomerase and housekeeping gene expression were found in positive control group (colon cancer tissue) and sample tissue.Conclusions:The absence of telomerase gene expression in HUVECs might be due to the mortality of these cells or the low level of telomerase gene expression in these cells under normal circumstances.

Two cases of Oto palate digital syndrome type II: Clinical features and a study of telomere length maintenance pathways.

Objective: To study the clinicopathological features of Oto palato digital syndrome type II (OPD-II) and telomere length maintenance pathway. Design: Case-control study of OPD-II. Setting: Different clinicopathological studies along with telomere length maintenance pathways were investigated. Participants: Clinically diagnosed two OPD-II patients and four randomly selected age-matched normal individual. Main outcome measures: Studied OstiumSecondum Atrial Septal Defects (osASD) by ECG, testes image by USG, subcortical dysrythmia by EEG, image of corpus callosum by MRI, 17 α oH progesterone level in blood. telomere length, expression of telomerase and telomere-associated genes of PBMC were also measured. Results: Two children with OPD-II showed characteristic clinical symptoms such as cleft palate, broad forehead, facial dysmorphism, flat nasal bridge, low birth weight but no abnormality of the digits. The patient-1 exhibited osASD, bilateral undescended testes with hypospadiusand eventually developed seizure disorder in the follow up, ambiguous genitalia and high level of 17 α oH progesterone in blood. Interestingly, this patient showed shorter telomere length of PBMC – (~60%), low (

Regulation of the Human Telomerase Gene TERT by Telomere Position Effect-Over Long Distances (TPE-OLD): Implications for Aging and Cancer.

Telomerase is expressed in early human development and then becomes silenced in most normal tissues. Because ~90% of primary human tumors express telomerase and generally maintain very short telomeres, telomerase is carefully regulated, particularly in large, long-lived mammals. In the current report, we provide substantial evidence for a new regulatory control mechanism of the rate limiting catalytic protein component of telomerase (hTERT) that is determined by the length of telomeres. We document that normal, young human cells with long telomeres have a repressed hTERT epigenetic status (chromatin and DNA methylation), but the epigenetic status is altered when telomeres become short. The change in epigenetic status correlates with altered expression of TERT and genes near to TERT, indicating a change in chromatin. Furthermore, we identified a chromosome 5p telomere loop to a region near TERT in human cells with long telomeres that is disengaged with increased cell divisions as telomeres progressively shorten. Finally, we provide support for a role of the TRF2 protein, and possibly TERRA, in the telomere looping maintenance mechanism through interactions with interstitial TTAGGG repeats. This provides new insights into how the changes in genome structure during replicative aging result in an increased susceptibility to age-related diseases and cancer prior to the initiation of a DNA damage signal.