hTERT Epigenetics Provides New Perspectives for Diagnosis and Evidence-Based Guidance of Chemotherapy in Cancer.

Strong epigenetic pan-cancer biomarkers are required to meet several current, urgent clinical needs and to further improve the present chemotherapeutic standard. We have concentrated on the investigation of epigenetic alteration of the hTERT gene, which is frequently epigenetically dysregulated in a number of cancers in specific developmental stages. Distinct DNA methylation profiles were identified in our data on early urothelial cancer. An efficient EpihTERT assay could be developed utilizing suitable combinations with sequence-dependent thermodynamic parameters to distinguish between differentially methylated states. We infer from this data set, the epigenetic context, and the related literature that a CpG-rich, 2800 bp region, a prominent CpG island, surrounding the transcription start of the hTERT gene is the crucial epigenetic zone for the development of a potent biomarker. In order to accurately describe this region, we have named it "Acheron" (Ἀχέρων). In Greek mythology, this is the river of woe and misery and the path to the underworld. Exploitation of the DNA methylation profiles focused on this region, e.g., idiolocal normalized Methylation Specific PCR (IDLN-MSP), opens up a wide range of new possibilities for diagnosis, determination of prognosis, follow-up, and detection of residual disease. It may also have broad implications for the choice of chemotherapy.

From Lab Bench to Hope: Emerging Gene Therapies in Clinical Trials for Alzheimer's Disease.

Alzheimer's disease is a progressive neurodegenerative disorder that affects memory and cognitive abilities, affecting millions of people around the world. Current treatments focus on the management of symptoms, as no effective therapy has been approved to modify the underlying disease process. Gene therapy is a promising approach that can offer disease-modifying treatment for AD, targeting various aspects of the pathophysiology of the disease. This review presents a comprehensive overview of the current state of gene therapy research for AD, with a specific focus on clinical trials and preclinical studies that have used nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), apolipoprotein E2 (APOE2), and human telomerase reverse transcriptase (hTERT) as therapeutic gene therapy approaches. These gene targets have shown potential to alleviate the neuropathology of AD in animal studies and have demonstrated feasibility and safety in non-human primates. Despite the failure of the NGF gene therapy approach in clinical trials, we have reviewed and highlighted the reported findings and evaluations from the trials. Furthermore, the review included the conclusions of postmortem brain tissue analysis of AD patients who received NGF gene therapy. The goal is to learn from the failed trials and improve the approach in the future. Although gene therapy shows promise, it faces several challenges and limitations, including optimizing gene delivery methods, enhancing safety and efficacy profiles, and determining long-term results. This review contributes to the growing body of literature on innovative treatments for AD and highlights the need for more research and development to advance gene therapy as a viable treatment option for AD.

The Effects of Smoking on Telomere Length, Induction of Oncogenic Stress, and Chronic Inflammatory Responses Leading to Aging.

Telomeres, potential biomarkers of aging, are known to shorten with continued cigarette smoke exposure. In order to further investigate this process and its impact on cellular stress and inflammation, we used an in vitro model with cigarette smoke extract (CSE) and observed the downregulation of telomere stabilizing TRF2 and POT1 genes after CSE treatment. hTERT is a subunit of telomerase and a well-known oncogenic marker, which is overexpressed in over 85% of cancers and may contribute to lung cancer development in smokers. We also observed an increase in hTERT and ISG15 expression levels after CSE treatment, as well as increased protein levels revealed by immunohistochemical staining in smokers' lung tissue samples compared to non-smokers. The effects of ISG15 overexpression were further studied by quantifying IFN-γ, an inflammatory protein induced by ISG15, which showed greater upregulation in smokers compared to non-smokers. Similar changes in gene expression patterns for TRF2, POT1, hTERT, and ISG15 were observed in blood and buccal swab samples from smokers compared to non-smokers. The results from this study provide insight into the mechanisms behind smoking causing telomere shortening and how this may contribute to the induction of inflammation and/or tumorigenesis, which may lead to comorbidities in smokers.

Enhancing Proliferation of Stem Cells from Human Exfoliated Deciduous Teeth (SHED) through hTERT Expression while Preserving Stemness and Multipotency.

BACKGROUND: Stem cells from human exfoliated deciduous teeth (SHED) hold promise in regenerative medicine owing to their multipotent capabilities resembling mesenchymal stem cells (MSCs). Despite their potential, SHED have not been extensively investigated because their limited lifespan and unavailability of cell-lines pose challenges for therapeutic applications. This study investigated the effect of ectopic human telomerase reverse transcriptase (hTERT) expression on SHEDs' proliferation while preserving stemness and genomic integrity. METHODS: Deciduous teeth were collected from children aged 6-10 years. After isolation and characterization, the SHED were transduced with pBabe-puro-hTERT retrovirus to establish SHED cell-line, which was evaluated and compared with pBabe-puro (mock control) for stemness, multipotency and growth attributes through flow cytometry, trilineage differentiation, and growth kinetics. We also estimated hTERT gene expression, genomic integrity, and validated cell-line through STR analysis. RESULTS: Following hTERT transduction, SHED displayed elevated hTERT gene expression while retaining fibroblast-like morphology and mesenchymal stem cell markers. Moreover, after hTERT transduction cellular shape remained same along with increased replicative lifespan and proliferation potential. SHED-hTERT cells exhibited multi-potency and maintained stemness, as evidenced by surface marker expression and multilineage differentiation. Furthermore, genomic integrity was not affected by hTERT integration, as confirmed by STR analysis and CDKN2A gene assessment. CONCLUSION: Ectopic hTERT expression in SHED successfully prolonged their replicative lifespan and improved their ability to proliferate and migrate, while preserving their stemness, multipotency and genomic integrity, suggesting minimal carcinogenic risk. Establishment of SHED cell-line holds potential in regenerative medicine applications, especially in cell-based drugs and tissue engineering experiments.

Development and validation of novel keloid-derived immortalized fibroblast cell lines.

Keloids are a common connective tissue disorder with an ill-understood etiopathogenesis and no effective treatment. This is exacerbated because of the absence of an animal model. Patient-derived primary keloid cells are insufficient as they age through passaging and have a limited supply. Therefore, there is an unmet need for development of a cellular model that can consistently and faithfully represent keloid's pathognomic features. In view of this, we developed keloid-derived immortalized fibroblast (KDIF) cell lines from primary keloid fibroblasts (PKF) by transfecting the human telomerase reverse transcriptase (hTERT) gene. The TERT gene encodes the catalytic subunit of the telomerase enzyme, which is responsible for maintaining the cellular replicative potential (cellular immortalization). Primary fibroblasts from keloid-specific lesional (peripheral, middle, and top) as well as extralesional sites were isolated and evaluated for cell line development and comparative cellular characteristics by employing qRT-PCR and immunofluorescence staining. Moreover, the immortalized behavior of KDIF cell lines was evaluated by comparing with cutaneous fibrosarcoma and dermatofibrosarcoma protuberans cell lines. Stable KDIF cell lines with elevated expression of hTERT exhibited the cellular characteristics of site-specific keloid fibroblasts. Histochemical staining for ß-galactosidase revealed a significantly lower number of ß-gal-positive cells in all three KDIF cell lines compared with that in PKFs. The cell growth curve pattern was studied over 10 passages for all three KDIF cell lines and was compared with the control groups. The results showed that all three KDIF cell lines grew significantly faster and obtained a fast growing characteristic as compared to primary keloid and normal fibroblasts. Phenotypic behavior in growth potential is an indication of hTERT-mediated immortalized transformation. Cell migration analysis revealed that the top and middle KDIF cell lines exhibited similar migration trend as site-specific PKFs. Notably, peripheral KDIF cell line showed significantly enhanced cell migration in comparison to the primary peripheral fibroblasts. All KDIF cell lines expressed Collagen I protein as a keloid-associated fibrotic marker. Functional testing with triamcinolone inhibited cell migration in KDIF. ATCC short tandem repeat profiling validated the KDIF as keloid representative cell line. In summary, we provide the first novel KDIF cell lines. These cell lines overcome the limitations related to primary cell passaging and tissue supply due to immortalized features and present an accessible and consistent experimental model for keloid research.

DDB2 promotes melanoma cell growth by transcriptionally regulating the expression of KMT2A and predicts a poor prognosis.

Identification of potential key targets of melanoma, a fatal skin malignancy, is critical to the development of new cancer therapies. Lysine methyltransferase 2A (KMT2A) promotes melanoma growth by activating the human telomerase reverse transcriptase (hTERT) signaling pathway; however, the exact mechanism remains elusive. This study aimed to reveal new molecular targets that regulate KMT2A expression and melanoma growth. Using biotin-streptavidin-agarose pull-down and proteomics, we identified Damage-specific DNA-binding protein 2 (DDB2) as a KMT2A promoter-binding protein in melanoma cells and validated its role as a regulator of KMT2A/hTERT signaling. DDB2 knockdown inhibited the expression of KMT2A and hTERT and inhibited the growth of melanoma cells in vitro. Conversely, overexpression of DDB2 activated the expression of KMT2A and promoted the growth of melanoma cells. Additionally, we demonstrated that DDB2 expression was higher in tumor tissues of patients with melanoma than in corresponding normal tissues and was positively correlated with KMT2A expression. Kaplan-Meier analysis showed a poor prognosis in patients with high levels of DDB2 and KMT2A. Overall, our data suggest that DDB2 promotes melanoma cell growth through the transcriptional regulation of KMT2A expression and predicts poor prognosis. Therefore, targeting DDB2 may regulate the effects of KMT2A on melanoma growth and progression, providing a new potential therapeutic strategy for melanoma.

Dual­regulated oncolytic adenovirus carrying ERCC1­siRNA gene possesses potent antitumor effect on ovarian cancer cells.

Ovarian cancer is a multifactorial and deadly disease. Despite significant advancements in ovarian cancer therapy, its incidence is on the rise and the molecular mechanisms underlying ovarian cancer invasiveness, metastasis and drug resistance remain largely elusive, resulting in poor prognosis. Oncolytic viruses armed with therapeutic transgenes of interest offer an attractive alternative to chemical drugs, which often face innate and acquired drug resistance. The present study constructed a novel oncolytic adenovirus carrying ERCC1 short interfering (si)RNA, regulated by hTERT and HIF promoters, termed Ad­siERCC1. The findings demonstrated that this oncolytic adenovirus effectively inhibits the proliferation, migration and invasion of ovarian cancer cells. Furthermore, the downregulation of ERCC1 expression by siRNA ameliorates drug resistance to cisplatin (DDP) chemotherapy. It was found that Ad­siERCC1 blocks the cell cycle in the G1 phase and enhances apoptosis through the PI3K/AKT­caspase­3 signaling pathways in SKOV3 cells. The results of the present study highlighted the critical effect of oncolytic virus Ad­siERCC1 in inhibiting the survival of ovarian cancer cells and increasing chemotherapy sensitivity to DDP. These findings underscore the potent antitumor effect of Ad­siERCC1 on ovarian cancers in vivo.

Effects of metformin and silodosin as supplementary treatments to abiraterone on human telomerase reverse transcriptase (hTERT) level in metastatic castration-resistant prostate cancer (mCRPC) cells: An in vitro study.

The antiproliferative properties of metformin and silodosin have been observed in prostate cancer. Furthermore, it is hypothesized that the molecular pathways related to these drugs may impact the levels of human telomerase reverse transcriptase (hTERT) in prostate cancer cells. The aim of this study was to assess the effect of metformin and silodosin on the levels of hTERT in metastatic castration-resistant prostate cancer (mCRPC) cells. The present study employed an experimental design with a post-test-only control group. This study utilized the PC3 cell line as a model for mCRPC. A viability experiment was conducted using the CCK-8 method to determine the inhibitory concentration (IC50) values of metformin, silodosin, and abiraterone acetate (AA) after a 72-hour incubation period of PC3 cells. In order to investigate the levels of hTERT, PC3 cells were divided into two control groups: a negative control and a standard therapy with AA. Additionally, three experimental combination groups were added: metformin with AA; silodosin with AA; and metformin, silodosin and AA. The level of hTERT was measured using sandwich ELISA technique. The difference in hTERT levels was assessed using ANOVA followed by a post hoc test. The IC50 values for metformin, silodosin, and AA were 17.7 mM, 44.162 mM, and 66.9 µM, respectively. Our data indicated that the combination of metformin with AA and the combination of metformin, silodosin and AA decreased the hTERT levels when compared to control, AA, and silodosin with AA. The administration of metformin resulted in a reduction of hTERT levels in the PC3 cell line, but the impact of silodosin on hTERT levels was not statistically significant compared to AA group.

hTERT-Immortalized Mesenchymal Stem Cell-Derived Extracellular Vesicles: Large-Scale Manufacturing, Cargo Profiling, and Functional Effects in Retinal Epithelial Cells.

As the economic burden associated with vision loss and ocular damage continues to rise, there is a need to explore novel treatment strategies. Extracellular vesicles (EVs) are enriched with various biological cargo, and there is abundant literature supporting the reparative and immunomodulatory properties of stem cell EVs across a broad range of pathologies. However, one area that requires further attention is the reparative effects of stem cell EVs in the context of ocular damage. Additionally, most of the literature focuses on EVs isolated from primary stem cells; the use of EVs isolated from human telomerase reverse transcriptase (hTERT)-immortalized stem cells has not been thoroughly examined. Using our large-scale EV-manufacturing platform, we reproducibly manufactured EVs from hTERT-immortalized mesenchymal stem cells (MSCs) and employed various methods to characterize and profile their associated cargo. We also utilized well-established cell-based assays to compare the effects of these EVs on both healthy and damaged retinal pigment epithelial cells. To the best of our knowledge, this is the first study to establish proof of concept for reproducible, large-scale manufacturing of hTERT-immortalized MSC EVs and to investigate their potential reparative properties against damaged retinal cells. The results from our studies confirm that hTERT-immortalized MSC EVs exert reparative effects in vitro that are similar to those observed in primary MSC EVs. Therefore, hTERT-immortalized MSCs may represent a more consistent and reproducible platform than primary MSCs for generating EVs with therapeutic potential.

Noncanonical functions of telomerase and telomeres in viruses-associated cancer.

The cause of cancer is attributed to the uncontrolled growth and proliferation of cells resulting from genetic changes and alterations in cell behavior, a phenomenon known as epigenetics. Telomeres, protective caps on the ends of chromosomes, regulate both cellular aging and cancer formation. In most cancers, telomerase is upregulated, with the telomerase reverse transcriptase (TERT) enzyme and telomerase RNA component (TERC) RNA element contributing to the maintenance of telomere length. Additionally, it is noteworthy that two viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV), utilize telomerase for their replication or persistence in infected cells. Also, TERT and TERC may play major roles in cancer not related to telomere biology. They are involved in the regulation of gene expression, signal transduction pathways, cellular metabolism, or even immune response modulation. Furthermore, the crosstalk between TERT, TERC, RNA-binding proteins, and microRNAs contributes to a greater extent to cancer biology. To understand the multifaceted roles played by TERT and TERC in cancer and viral life cycles, and then to develop effective therapeutic strategies against these diseases, are fundamental for this goal. By investigating deeply, the complicated mechanisms and relationships between TERT and TERC, scientists will open the doors to new therapies. In its analysis, the review emphasizes the significance of gaining insight into the multifaceted roles that TERT and TERC play in cancer pathogenesis, as well as their involvement in the viral life cycle for designing effective anticancer therapy approaches.

CRL4DCAF4 E3 ligase-mediated degradation of MEN1 transcriptionally reactivates hTERT to sustain immortalization in colorectal cancer cells.

Telomerase reactivation is implicated in approximately 85% of human cancers, yet its underlying mechanism remains elusive. In this study, we elucidate that the Cullin RING Ubiquitin Ligase 4 (CRL4) complex drives the reactivation of human telomerase reverse transcriptase (hTERT) in colorectal cancer (CRC) by degrading the tumor suppressor, menin 1 (MEN1). Our data show that, in noncancerous intestinal epithelial cells, the transcription factor specificity protein 1 (Sp1) recruits both the histone acetyltransferase p300 and MEN1 to suppress hTERT expression, thus maintaining telomere shortness post-cell division. Inflammation-induced microenvironments trigger an activation of the CRL4DCAF4 E3 ligase, leading to MEN1 ubiquitination and degradation in CRC cells. This process nullifies MEN1's inhibitory action, reactivates hTERT expression at the transcriptional level, interrupts telomere shortening, and spurs uncontrolled cellular proliferation. Notably, MEN1 overexpression in CRC cells partially counteracts these oncogenic phenotypes. NSC1517, an inhibitor of the CRL4DCAF4 complex identified through high-throughput screening from a plant-derived chemical pool, hinders MEN1 degradation, attenuates hTERT expression, and suppresses tumor growth in mouse xenograft models. Collectively, our research elucidates the transcriptional mechanism driving hTERT reactivation in CRC. Targeting the CRL4DCAF4 E3 ligase emerges as a promising strategy to counteract cancer cell immortalization and curb tumor progression.

A phase 1 trial of human telomerase reverse transcriptase (hTERT) vaccination combined with therapeutic strategies to control immune-suppressor mechanisms.

The presence of inhibitory immune cells and difficulty in generating activated effector T cells remain obstacles to development of effective cancer vaccines. We designed a vaccine regimen combining human telomerase reverse transcriptase (hTERT) peptides with concomitant therapies targeting regulatory T cells (Tregs) and cyclooxygenase-2 (COX2)-mediated immunosuppression. This Phase 1 trial combined an hTERT-derived 7-peptide library, selected to ensure presentation by both HLA class-I and class-II in 90% of patients, with oral low-dose cyclophosphamide (to modulate Tregs) and the COX2 inhibitor celecoxib. Adjuvants were Montanide and topical TLR-7 agonist, to optimise antigen presentation. The primary objective was determination of the safety and tolerability of this combination therapy, with anti-cancer activity, immune response and detection of antigen-specific T cells as additional endpoints. Twenty-nine patients with advanced solid tumours were treated. All were multiply-pretreated, and the majority had either colorectal or prostate cancer. The most common adverse events were injection-site reactions, fatigue and nausea. Median progression-free survival was 9 weeks, with no complete or partial responses, but 24% remained progression-free for ≥6 months. Immunophenotyping showed post-vaccination expansion of CD4+ and CD8+ T cells with effector phenotypes. The in vitro re-challenge of T cells with hTERT peptides, TCR sequencing, and TCR similarity index analysis demonstrated the expansion following vaccination of oligoclonal T cells with specificity for hTERT. However, a population of exhausted PD-1+ cytotoxic T cells was also expanded in vaccinated patients. This vaccine combination regimen was safe and associated with antigen-specific immunological responses. Clinical activity could be improved in future by combination with anti-PD1 checkpoint inhibition to address the emergence of an exhausted T cell population.

Telomere length and TERT polymorphisms as biomarkers in asbestos-related diseases.

BACKGROUND: Asbestos exposure has been proposed as a risk factor for shorter telomere length. The aim of our study was to investigate whether telomere length in leukocytes and hTERT genetic polymorphisms may serve as potential biomarkers for the risk of developing asbestos-related diseases and as biomarkers of progression and chemotherapy response rate in malignant mesothelioma (MM). SUBJECTS AND METHODS: We conducted two retrospective studies. In the first study, a case-control study, telomere length and hTERT polymorphisms were determined in patients with MM, subjects with pleural plaques and controls without the asbestos related disease, who were occupationally exposed to asbestos. In the second study, a longitudinal observational study, telomere length was also determined in samples from MM patients before and after chemotherapy. Telomere length was determined by monochromatic multiplex quantitative polymerase chain reaction (PCR), while competitive allele-specific PCR was used to genotype hTERT rs10069690, rs2736100 and rs2736098. Logistic regression and survival analysis were used in statistical analysis. RESULTS: Patients with MM had shorter telomere length than subjects with pleural plaques (p < 0.001). After adjustment for age, rs2736098 CT, and rs10069690 TT and CT+TT genotypes were significantly associated with a higher risk of MM (padj = 0.023; padj = 0.026 and padj = 0.017), while rs2736100 AA and CA+AA genotypes conferred to a lower risk for MM compared to all other subjects (padj = 0.017, and padj = 0.026). Telomere length was not associated with a response to chemotherapy (p > 0.05) or time to disease progression (p > 0.05). Carriers of one or two polymorphic rs10069690 T alleles had a good response to chemotherapy (p = 0.039, and p = 0.048), these associations remained statistically significant after adjustment for age (padj = 0.019; padj = 0.017). Carriers of two polymorphic rs2736100 A alleles had a longer time to disease progression (p = 0.038). CONCLUSIONS: Shorter telomere length and hTERT polymorphisms may serve as a biomarker for the risk of developing MM. Additionally, rs10069690 and rs2736100 polymorphisms, but not telomere length, were associated with a chemotherapy response or MM progression.

Mechanistic influence of discreet conformation of human telomerase linker region.

Approximately 90% of malignancies have been shown to have human telomerase activity, establishing it as a viable therapeutic target. The crystal structure of telomerase was determined recently. However, the tertiary structure of the non-conserved flexible linker region remains unresolved. This study aims to predict the full-length tertiary structure of the human telomerase reverse transcriptase (hTERT). Two strategies were employed to determine the full-length structure of hTERT (1132 amino acids); iterative threading and a conjoined model generated from machine learning and energy functions. After energy minimization, Ramachandran Plot analysis, and simulation; the conjoined model was considered of better quality and stability. The linker region of the conjoined depicted two helices from approximately 275-284 and 201-211 amino acids respectively in contrast to the iterative threading model which has a single helix. Moreover, the region was observed to undergo major structural changes throughout the simulation. These changes signify its flexibility which might be due to the region having a significant number of glycine and proline and could enhance the clamping movement.Communicated by Ramaswamy H. Sarma.

Association of hTERT Gene Polymorphism and Colorectal Cancer (CRC) Risk in the Chinese Han Population.

The catalytic subunit telomerase reverse transcriptase (hTERT) is a prerequisite for malignant transformation of human cells. Colorectal cancer (CRC) is a common malignant tumor. The genetic association of hTERT gene rs2853669 and rs2736098 polymorphisms with CRC was surveyed in the Chinese population. Two hundreds patients with CRC and 200 healthy controls were taken for blood sample collection. Sanger sequencing was applied for genotyping. Multiple logistic regression analysis was performed, and odds ratio (OR) together with confidence interval (CI) were calculated to obtain the corresponding association power. Among CRC cases (49.50%), hTERT gene rs2736098 GA genotype carriers were more prevalent compared with the control group (41.00%, P = 0.035), which increased the risk of CRC by 1.576 times (95% CI, 1.031-2.409). Distribution of the rs2736098 genotypes was significantly associated with TNM stage, tumor differentiation, tumor size and lymph node metastasis (P < 0.05). The frequencies of hTERT gene rs2853669 polymorphism were not significantly different between CRC patients and healthy controls. Logistic regression analysis indicated that both body mass index (BMI) and hTERT gene rs2736098 polymorphism remained significantly correlated with CRC susceptibility. The frequencies of hTERT gene rs2853669 polymorphism did not differ significantly between CRC patients and control group (P > 0.05). The hTERT gene rs2736098 polymorphism was correlated with CRC risk in the Chinese Han population, and the GA genotype was a risk element for the onset of CRC.

hTERT Gene Modification Using CRISPR-dCas9-dnmt3a System as a Therapeutic Approach Against Glioma.

Background: Abnormal DNA methylation patterns have been reported in various diseases, including different cancers. CRISPR/Cas9 is a low-cost and highly effective gene editing tool that has lately revolutionized biotechnology. Studies have shown that the CRISPR/Cas9 system can effectively target and correct methylation. Objectives: Telomerase plays a survival role for cancer cells. It is encoded by the hTERT gene. The effectiveness of CRISPR/Cas9 in targeting hTERT to treat glioma cancer cells was assessed in this study. Methods: EF1a-hsaCas9-U6-gRNA vector carrying sgRNA and Cas9 hybrids were used to transfect U87 glioma cells. Four and eight µg/mL polybrene concentrations were investigated to improve transfection efficiency. The expression level of hTERT that has undergone metabisulfite modification was assessed using real-time PCR. Flow cytometry and Western blotting were also used to determine whether telomerase was present in the cells. High-resolution melting analysis (HRM) was used to examine the hTERT promoter's methylation. Finally, flow cytometry was used to measure the apoptotic rate of transfected U87 cells. Results: The findings demonstrated that gRNA significantly boosted transfection effectiveness. Significant variations were seen in the expression of hTERT in U87 cells at 4 µg/mL polybrene and 80 µg/mL transfection compared to transfection without gRNA and basal cells. Flow cytometry showed a decrease in hTERT levels in transfected cells. Furthermore, transfection with gRNA increased U87 cell apoptosis compared to transfection without gRNA. Conclusions: It appears that the designed CRISPR/Cas9 system can reduce hTERT expression and telomerase activity and thus inhibit glioma cell growth.

Efficacy of Bee Products (Anzer Honey, Pollen and Propolis) in Detection and Healing of Damage Induced by Antidiabetic Drug Vildagliptin/Metformin Hydrochloride in Healthy Human Pancreatic Cells: Cytotoxic, Genotoxic and Biochemical Studies.

BACKGROUND AND OBJECTIVE: Although drugs are powerful therapeutic agents, they have a range of side effects. These side effects are sometimes cellular and not clinically noticeable. Vildagliptin/metformin hydrochloride is one of the most widely used oral antidiabetic drugs with two active ingredients. In this study, we investigated its harmful effects on the metabolic activation system in healthy human pancreatic cells "hTERT-HPNE", and we aimed to improve these harmful effects by natural products. To benefit from the healing effect, we used the unique natural products produced by the bees of the Anzer Plateau in the Eastern Black Sea Region of Turkey. METHODS: Cytotoxic and genotoxic effects of the drug were investigated by different tests, such as MTT, flow cytometry-apoptosis and comet assays. Anzer honey, pollen and propolis were analyzed by gas chromatography/mass spectrometry (G/C-MS). A total of 19 compounds were detected, constituting 99.9% of the samples. RESULTS: The decrease in cell viability at all drug concentrations was statistically significant compared to the negative control (P<0.05). A statistically significant decrease was detected in the apoptosis caused by vildagliptin/metformin hydrochloride with the supplementation of Anzer honey, pollen and propolis in hTERT-HPNE cells (P<0.05). CONCLUSION: This study can contribute to other studies testing the healing properties of natural products against the side effects of oral antidiabetics in human cells. In particular, Anzer honey, pollen and propolis can be used as additional foods to maintain cell viability and improve heal damage and can be evaluated against side effects in other drug studies.

Telomerase Expression Related with Poor Immune Response to HCV Core Antigen in Egyptian HCV Patients' PBMCs.

Background: Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. Aim: This study aimed to assess serum human telomerase enzyme (hTERT) levels and their relation to the progression of liver disease. Also, it aimed to assess the effect of hepatitis C virus (HCV) core protein on memory T-cells in HCV patients with or without HCC and the correlation between memory cell phenotype and the progression of the disease in the same patients. Methods: HTERT level in serum was assessed through relative quantitative RT-PCR. Flow cytometric analysis was used to assess T-cell responsiveness (as IFN- γ secretion) before and after stimulation with HCV core protein and the memory CD8+ cell phenotype using several differentiation markers. Results: HTERT was found to be increased in a stepwise manner upon comparing its level in controls, chronic hepatitis patients, cirrhotic patients, and HCC patients. T-cells showed a similar manner of stepwise decrease in response (decreased IFN- γ secretion) in HCC patients compared to HCV patients without HCC and controls. Also, late differentiated memory cells (CD8+, CD27-, CD28-, CD45RA+, and CCR7-) were depleted in HCC patients compared to HCV patients without HCC. Conclusion: These results suggest a negative correlation between hTERT and IFN- γ secretion by T-cells in HCV patients and that this relationship, along with the depletion of late differentiated memory cells, could help the progression of liver disease to HCC.

Differential Expression of the hTERT Gene in Umbilical Cord-Derived Mesenchymal Stem Cells Cocultured with B Cell Precursor Leukemia Cell Microparticles or CD41+/CD61+ Platelet Microparticles.

Several investigations are being done to increase the short lifetime of mesenchymal stem cells (MSCs). One of the crucial genes involved in the immortalization of MSCs, hTERT (human telomerase reverse transcriptase), is activated in most publications using viral-based techniques. In this work, we investigated the use of platelet-derived (PMPs) and B cell precursor leukemia-derived microparticles as a nonviral method to trigger and compare the expression of the hTERT gene in MSCs. MSCs were extracted from the umbilical cord for the current investigation and identified using a flow cytometry approach and an inverted microscope. The Nalm-6 cell line and platelet concentrate were used to isolate microparticles (MPs). MSCs and MPs were cocultured for 14 days at 25-, 50-, and 100 µg/ml concentrations. qRT-PCR was used to research the expression of the hTERT gene. SPSS 26.0's t test was used to compare the outcomes. After coculture with platelet MPs, MSCs had higher levels of hTERT gene expression than the control group. In contrast, this gene's expression was concurrently decreased in MSCs exposed to MPs generated from Nalm-6. We demonstrated that following 14-day treatment, PMP significantly boosted the hTERT gene expression in MSCs, while the Nalm-6 MPs lowered the gene expression. However, additional studies are necessary due to the stability of hTERT gene expression and the immortalization of MSCs following exposure.

Behaviour of hTERT in the tears of neophyte contact lens wearers during the sleep/wake cycle.

PURPOSE: To investigate the behaviour of telomerase reverse transcriptase (hTERT) in the tears of healthy neophyte contact lenses-wearing individuals during the sleep/wake cycle. A subsequent aim was to investigate whether hTERT behaviour was associated with inflammatory mediators interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in tears. METHODS: Flush tears were collected from 19 healthy, non-contact lens-wearing participants (11 males, 8 females, mean age 31.9 ± 5.7 years), before and during contact lens wear. Tears were collected at noon, before sleep and upon awakening and levels of hTERT, IL-6 and TNF-α, were determined using enzyme-linked immunosorbent assays (ELISA). RESULTS: hTERT levels (median [interquartile range]) during contact lens wear were significantly higher before sleep (436.5 (263.9 - 697.7) ng/ml compared to the same time point without contact lenses (256.1 (0.0 - 590.9) ng/ml (p = 0.01). There was no difference between contact lens wear (851.3 [353.2 - 2109.9]) ng/ml, and no wear (1091.0 [492.3 - 3045.4]) ng/ml, upon awakening (p = 0.94). A significant increase was found upon awakening compared to before sleep, irrespective of the presence of a contact lens (p = 0.02). IL-6 and TNF-α levels in tears were below the limit of detection. CONCLUSIONS: The study showed that hTERT increases after a contact lens is placed on the eye, but this change is small, compared to the impact of overnight eye closure. Taken together with the lack of responses of the inflammatory markers monitored at the same time points, this may suggest that hTERT can respond both to low-level stress stimuli acting on the ocular surface, and to situations where inflammation is a likely factor.